ERD

Examine.com

Research Digest

Kamal Patel

5 Year Anniversary Edition

1

From the Editor

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2

Table of Contents

Something fishy: How a component of fish oil may counteract

05
the effects of some chemotherapy
Fish oil isnt necessarily benign ... it turns out that certain fatty acids might
partially negate chemotherapy

13

The study that didnt end the low-fat/low-carb diet wars

24

Investigating vitamin D as a performance enhancer

32

Not-so-safe supplements

41

The espresso effect: caffeine and circadian rhythm

A recent metabolic ward study set the low-carb world on fire, and produced
many inaccurate media headlines disparaging low-carb diets. We cover the
study and its implications, detail by detail.

Having sufficient vitamin D levels has been associated with better muscle
recovery. This trial not only looks at the question of causality, but also
addresses some potential mechanisms of vitamin Ds benefit for exercise

Studies have shown that supplement buyers generally trust the supplements
they buy. That might not be the safest assumption, as dietary supplements
that are presumed helpful or neutral may sometimes cause serious side
effects, as quantified by this study.

Your daily rhythms are influenced by zeitgebers such as light and exercise.
But until now, we havent known the exact impact of late-day caffeine intake
on melatonin and circadian rhythms.

Contributors
Researchers

Margaret Wertheim
M.S., RD

Alex Leaf
M.S(c)

Courtney Silverthorn Zach Bohannan

Ph.D.
M.S.

Anders Nedergaard
Ph.D.

Jeff Rothschild
M.Sc., RD

Katherine Rizzone
M.D.

Spencer Nadolsky
D.O.

Greg Palcziewski
Ph.D. (c)

Editors

Gregory Lopez
Pharm.D.

Reviewers

Pablo Sanchez Soria Kamal Patel

Ph.D.
M.B.A., M.P.H.,
Ph.D(c)

Arya Sharma
Ph.D., M.D.

Natalie Muth
M.D., M.P.H., RD

Stephan Guyenet
Ph.D.

Mark Kern
Ph.D., RD

Gillian Mandich
Ph.D(c)

Adel Moussa
Ph.D(c)

Sarah Ballantyne
Ph.D.

Something fishy: How

a component of fish
oil may counteract
the effects of some
chemotherapy
Increased Plasma Levels of
Chemoresistance-Inducing Fatty
Acid 16:4(n-3) After Consumption
of Fish and Fish Oil

Introduction
Cancer is an incredibly broad group of diseases characterized by similar features, the most notable being
uncontrolled cell growth. Although researchers have
made great strides in understanding the molecular
mechanisms behind various cancers, they are still
working on creating effective therapies that specifically target these mechanisms. This means that many
therapies rely on relatively old-fashioned treatments:
chemo or radiation, which kill the tumor cells faster or
more effectively than they kill normal cells. As might
be expected from such harsh therapies for such serious
diseases, many of these cancer therapies are associated
with severe side effects.
To address these side effects (and possibly as a result of
newfound interest in their health), many patients turn
to dietary remedies, including a variety of supplements.
Fish oil is one of the most popular choices, and it is

benefits of fish oil supplementation and oily fish consumption have been widely researched for decades,
scientists are now beginning to assess the components
of fish oil in more detail, especially as they pertain to
specific populations or interactions with medications.
The group who conducted this study was the first group
to identify certain fatty acids called platinum-induced
fatty acids (PIFAs) that can induce resistance to chemotherapy in mice. Specifically, they identified 12S-HHT
and 16:4(n-3) as two fatty acids that can cause resistance to chemotherapy by altering DNA damage repair
mechanisms. Figure 1 depicts how PIFAs may interact
with macrophages to ultimately induce some level of
chemoresistance. This study is a follow-up to the previous mouse-based report and aims to examine the fish
oil supplementation habits of cancer patients, as well as
further clarify the effects of fish oil supplementation on
chemotherapy resistance.

used by an estimated 20% of cancer patients. However,

relatively little work has been done to assess fish oils

Fish oil supplementation is relatively common in

interactions with common cancer treatments like

cancer patients. The researchers conducting this

chemotherapy.

study recently identified certain components of fish

oil (especially platinum-induced fatty acid 16:4)

Fish oil is a common supplement, but its sources and

that can promote chemotherapy resistance in mice,

processing can vary greatly. It can come from any oily

so they sought to understand whether these effects

fish, including eel, herring, and mackerel. The specific

could also be seen in human tumors.

fatty acids components in fish oil can vary, depending

on the species and diet of the source fish. Since the

Figure 1: Possible mechanism for PIFA-induced chemoresistance

Reference: Houthuijzen et al. Nat Commun. 2014 Nov.

How fatty acids are named

BASIC FATTY ACID STRUCTURE

A NUMBERS GAME

Fatty acids are primarily long chains of carbon

and hydrogen atoms strung together at the end
of a carboxyl group (COOH), which is why they are
called acids. The first level of complexity among
fatty acids is their differences in length. This is what
researchers are referencing when they discuss shortchain and medium-chain fatty acids.

The naming system described above is common,

but many scientists rely on a more complete lipid
number naming convention. For example, the fatty
acid discussed in this paper, 16:4(n-3), is a 16-carbon fatty acid chain with four double bonds and a
double bond at the third carbon (making it a type of
omega-3 fatty acid). A depiction is shown in Figure 2.

GETTING TURNED AROUND

GETTING CONFUSED

The second level of complexity among fatty acids is

the introduction of double bonds between carbon
atoms, which produces a kink in the fatty acid
chain. Saturated fats dont have any double bonds,
monounsaturated fats have one, and polyunsaturated fats have many. An omega-3 fatty acid or n-3 is
a fatty acid with a double bond on the third carbon
of the chain.

Aside from the lipid number system, there are also

systematic names, which follow organic chemistry
naming conventions and turn out tongue-twisters
like hexadeca-4,7,10,13-tetraenoic acid, which is the
chemical name for the 16:4(n-3) acid mentioned
above. Then theres the trivial naming system that
follows historical names. For example arachidonic
acid is widely used but doesnt actually describe
any of the features of the fatty acid.

Figure 2: 16:4(n-3) fatty acid

Who and what was studied?

The design of this study is somewhat different than
most clinical studies because it assesses supplementation habits that could negatively affect life-saving
therapies. It would be incredibly unethical to treat
chemotherapy patients with fish oil and assess whether
or not their disease progressed, so instead, the study
authors assessed many of the most important aspects of
fish oils interaction with chemotherapy without actually conducting a clinical trial.
As a first step, the investigators tried to understand

whether consuming fish oil or fatty fish actually

increased PIFA levels in healthy volunteers. Thirty
healthy volunteers who had not recently consumed
fatty fish or fish oil were given either 10 or 50 milliliters
of fish oil from one of three different brands of commercially available fish oil (six in each group). Twenty
other healthy volunteers were fed 100 grams of either
tuna (a relatively lean fish that served as a sort of control), salmon, smoked mackerel, or cured herring (five
participants in each group). Blood was collected from
all participants before consumption as well as 1, 2, 4, 6,
8, and 24 hours after consumption of the fish oil or fish.
7

All of the supplements were also analyzed separately for

PIFA content.
To assess whether PIFAs could induce chemoresistance in human cancers, the researchers used a mouse
cancer model in which colon cancer cells are implanted under the skin of the mice, so that the researchers
can study the growth of the tumors. Once the tumors
reached a certain size, the researchers treated the mice
with a variety of chemotherapies, as well as a variety of
PIFA sources. These mice were either studied for tumor
growth over time or for pharmacodynamic studies
of the levels of chemotherapeutic and PIFA over time.
Similarly, the tumor-bearing mice were also treated
with purified eicosapentaenoic acid (EPA) to assess
whether mice can convert EPA into PIFAs and whether
those resultant PIFAs affected chemotherapy resistance.
Finally, to understand how patients actually use fish oil,
the researchers conducted a survey of over 400 patients,
to which only 118 patients responded. The questionnaire assessed whether or not patients used nutritional
supplements, which they used, and whether or not they
reported that use to their doctors.

It is difficult to study supplements that may minimize the effectiveness of lifesaving therapies. To do

[...] the
addition of
purified 16:4(n3) caused the
tumors to
grow at a rate
comparable to
that of tumors
from mice not
treated with
chemotherapy
at all.

so, the researchers studied whether fish oil or fish

consumption translated into increases in blood PIFA

controls effectively reduced cancer growth, but the

content in healthy volunteers, whether PIFA inges-

addition of purified 16:4(n-3) caused the tumors to

tion induces chemoresistance in a mouse model of

grow at a rate comparable to that of tumors from mice

tumor growth, and whether or not cancer patients

not treated with chemotherapy at all. This effect was

use fish oil supplements.

also seen when mice were treated with fish oil in doses equivalent to about three milliliters, or roughly 2.5

What were the findings?

The researchers found significant levels of 16:4(n-3)
fatty acids in all of the commercial fish oils they tested,
so they chose to focus the rest of their studies on that
specific fatty acid. In the mouse study, chemotherapy

grams, for a human. This is about double the dosage

recommended by the American Heart Association,
although even larger doses are often taken.
To assess the levels of fish oil required to induce chemoresistance, the researchers administered 100, 10, 1, and
0.1 microliters of fish oil to mice and assessed tumor
8

growth. Fish oil supplementation as low as one micro-

fish, relative to the amount that existed in the fish or oil

liter (again, equivalent to about a three milliliter dose

itself.

for humans) showed a significant reduction in chemotherapy effectiveness. Finally, when the researchers

The patient questionnaire revealed that 30% of patients

administered EPA to mice with tumors and assessed

regularly used nutritional supplements, and 11% reg-

both their serum 16:4(n-3) content as well as its ability

ularly used fish oil or other supplements containing

to induce chemoresistance, they found both elevated

omega-3 fatty acids. Eleven of the 13 patients (85%)

16:4(n-3) and tumor sizes similar to untreated tumors

who regularly used these supplements continued to

in mice that received EPA.

use them during therapy, but only six of them (55%)

reported their supplementation habits to their doctors.

In healthy volunteers, fish oil administration increased

16:4(n-3) levels in participant blood samples for up to
eight hours. It may have increased these levels for even
longer in some cases, but the trial only evaluated up
to eight hours. In the fish studies, participants who ate
mackerel or herring had increased levels of 16:4(n-3),
whereas participants who ate salmon had lower levels, and participants who ate tuna had levels similar to
baseline. As seen in Figure 3, fish oil increased human
blood levels of 16:4(n-3) to a greater degree than did

Fish oil and 16:4(n-3) administration induced tumor

chemoresistance in mouse models. Fish oil administration and oily fish consumption increased 16:4(n-3)
blood levels in healthy volunteers. 11% of cancer
patients used fish oil or omega-3 supplements, and
most of these patients used them during chemotherapy, with only about half reporting supplementation
to their doctor.

Figure 3: 16:4(n-3) levels in humans - higher than

expected with fish oil compared to fish

What does the study really

tell us?

Although the experiments in this study were somewhat

Although this study is unable to directly address

disparate, they do form the basis for a preliminary

whether or not fish oil supplementation reduces the

body of evidence. The study showed that fish oil and

effectiveness of chemotherapy in humans, it makes a

oily fish consumption could reduce the effectiveness

compelling case for chemotherapy patients to avoid fish

of chemotherapies in mice, that fish oil and oily fish

oil and oily fish consumption, at least in the days sur-

consumption in healthy volunteers increases blood

rounding chemotherapy treatment.

concentration of what is presumably the causative

patients often dont report fish oil usage to their doctors.

agent of this chemoresistance, and that a relatively large

Using a relatively standard tumor model that is known

percentage of cancer patients take dietary supplements,

to respond to chemotherapy, the researchers found

which include fish oil.

that 16:4(n-3) or fish oil administration reduces the

effectiveness of a variety of chemotherapies to the
point that the mouse tumors grew at the same rate as
untreated tumors. They observed similar effects from
purified EPA administration, which seems to indicate
that, at least in mice, EPA can be converted to 16:4(n-3)
through an unknown mechanism.
In humans, the researchers found that ingestion of fish
oil or oily fish increases levels of 16:4(n-3) in the blood

Added together, the experiments reported in this

paper provide a strong preliminary base of evidence
suggesting that cancer patients receiving chemotherapy may want to avoid taking fish oil supplements
around the time of their chemotherapy treatments.

The big picture

of healthy volunteers. Although this might seem like a

Chemotherapy is a balancing act. Since cancer is a dis-

relatively straightforward finding, it is critical to provide

ease state characterized by uncontrolled cell growth, it

evidence that human ingestion and digestion of fish oil

makes sense that targeting uncontrolled growth would

results in the presence of a specific fatty acid in the blood.

be a good therapeutic strategy. Unfortunately, there

arent any good ways to specifically target uncontrolled

Finally, the researchers found that most patients who

growth, so instead, chemotherapy targets rapid cell

use fish oil continue to do so during chemotherapy, and

growth. Although this makes it very good at killing

One example is St. Johns Wort,

which causes upregulation of enzymes
that process a variety of drugs,
including some chemotherapy agents.
10

cancer cells, it also results in off-target effects in other

rapidly dividing tissues, such as intestinal cells. This is
why many standard chemotherapeutic agents cause a
variety of gastrointestinal side effects, including vomiting and diarrhea. One of the key roles of oncologists
is to control chemotherapy doses to maximize cancer
cell death and minimize side effects. There are also a
wide array of supportive therapies available, such as
antiemetics, to help doctors control these side effects.
In many cases, cancer patients also seek out their own
supportive therapies, including supplements, in an
effort to enhance their quality of life while receiving
cancer treatment.
In most cases, it remains unclear whether supplements
are truly beneficial for cancer patients. In some other
cases, there is a clear contraindication for the use of
certain supplements while receiving certain chemotherapy agents. One example is St. Johns Wort, which
causes upregulation of enzymes that process a variety of
drugs, including some chemotherapy agents. However,
researchers have only recently begun studying interactions between chemotherapy and supplements, so there
are many other interactions that remain to be discovered.
This study represents a kind of miniature body of evidence to support the idea that 16:4(n-3) acids found
in fish oil may reduce the effectiveness of chemotherapy. Because cancer is such a lethal group of diseases
and because the therapies are already both carefully
calibrated and relatively arduous, it is critical to eliminate any agents or habits that could reduce therapeutic
effectiveness. Although all of the experiments in this
study were preliminary, they support the idea that fish
oil supplementation may have a negative impact on
chemotherapy effectiveness, thus indicating that supplementation management may be an important topic
for patients to discuss with a physician.
However, it is worth noting that most chemotherapy

[...] it may be
acceptable for
patients to take
fish oil or other
supplements
during the
times they are
not receiving
chemotherapy,
but further
study is needed
to confirm this
hypothesis.
effects they cause. Generally, chemotherapy is an intermittent treatment in which the patient receives chemo
drugs for a certain number of days and then takes a
break from the treatment in order to recover. This was
not really taken into account in the current study, so it
may be acceptable for patients to take fish oil or other
supplements during the times they are not receiving
chemotherapy, but further study is needed to confirm
this hypothesis.

regimens are not continuous, as a result of the side

11

Frequently Asked Questions

What alternative therapies or supplements are safe for

cancer patients?
This is a very broad and loaded question, and the
answer depends on the nature of a patients cancer.
Because cancers and their treatments are so diverse,
their interactions with supplements and alternative
therapies are similarly diverse. The most important
aspect of any supplementation or alternative therapy

regimen for cancer patients is constant and open com-

fatty acids may benefit prostate cancer. Some people

megadose fish oil in an attempt to quickly curb inflammation, and sometime encounter side effects such as
increased bleeding risk. So even if you deem yourself
healthy, it is always a good idea to consult with your
doctor before taking any supplements, and do research
into how supplements may interact with each other,
have different effects at different doses, etc. Theres even
a website to help you with that kind of thing!

munication with their oncologist or medical team. In

What should I know?

general, it is important for cancer patients to discuss

Fish oil supplementation and oily fish consumption may

any and all symptoms, medications, and dietary needs

negatively impact the effectiveness of chemotherapeutic

with their doctors. This is even more important in the

agents. The presumed causative factor of this interaction,

context of clinical trials, which often have very specific

16:4(n-3) PIFAs, reduced chemotherapeutic effectiveness

requirements and limitations.

in a mouse cancer model. These PIFAs are also present

in the bloodstream of healthy volunteers after fish oil

If the effects of fish oil are still not fully understood, is it

or oily fish consumption. Up to 30% of cancer patients

okay for healthy people to take it?

report using supplements, including fish oil. Based on

Generally speaking, fish oil is a relatively well-studied

this evidence, patients supplementing fish oil during

supplement in healthy populations. However, fish oil

chemotherapy should inform the oncologists treating

is not a commodity with one uniform composition

the cancer so they can provide appropriate advice.

among all products, and not all omega-3 supplements

are even fish oil in the first place.
As an example of how complicated things can get, recent
evidence points to possible prostate cancer harms
from certain fatty acids found in fish oil, while other

Almost everyone has had or knows someone who has

had cancer. Supplementation during chemotherapy is
rarely discussed to talk more about this issue, head
over to the Facebook ERD Forum.

Fish oil supplementation and oily fish

consumption may negatively impact
the effectiveness of chemotherapeutic
agents.
12

The study that didnt

end the low-fat/lowcarb diet wars
Calorie for Calorie, Dietary Fat
Restriction Results in More Body Fat
Loss than Carbohydrate Restriction
in People with Obesity

13

Introduction
Some of you may have already come across our blog
post about this recent blockbuster of a paper published
by Dr. Kevin Hall et al. If you have, stick around for our
extended F.A.Q. section where we tackle the numerous
questions brought up about the study. If you havent
read the blog, lets dive right into the trial analysis.
For some, the central dogma behind the hypothetical
superiority of low-carb diets for fat loss is the insulin
hypothesis of obesity. Part of this hypothesis states
that by restricting carbohydrates you will see a stepwise decrease in insulin secretions. Because insulin
plays a part in the regulation of fat storage, it has been
theorized that the less insulin secreted the more free
fatty acids will be released from adipose stores leading to increased fat oxidation and rapid fat loss. These
assumptions have led to the idea that low-carb diets
will induce greater fat loss over a low-fat diet even when
calories are held constant. Gary Taubes, an advocate
of the low-carb approach, posited the following in his
latest book, Why We Get Fat (p. 144-47):
any time we try to diet by any of the conventional
[low-fat] methods, and any time we decide to eat

healthy as its currently defined, we will remove the

most fattening carbohydrates from the diet and some
portion of total carbohydrates as well. And if we lose
fat, this will almost assuredly be the reason whyThis
is something that even researchers who run clinical
trials testing the effectiveness of different diets rarely
recognize.
Simply put, Taubes suggests that by reducing both
carbs and fat in low-fat diets it is possible that reductions in carbohydrate intake could be responsible for
any fat loss seen. Taubes is correct in that researchers
who run diet trials often alter the amount of fat and
carbohydrate participates eat, making it impossible
to determine if restricting one will lead to greater fat
loss over the other. Previous studies on low-fat and
low-carb diets have changed multiple variables simultaneously. So even though they end up comparing low-fat
and low-carb, they do not specifically reduce one macronutrient or the other from a baseline diet without
changing other variables. In the present study, Dr. Hall
and his team set out to eliminate that confounding
variable by subtracting either fat or carbs from the diet
without changing anything else. This was done under
tightly controlled conditions, to determine if indeed
there is a metabolic fat loss advantage to going low-carb.

[...] this was not a free living low-fat

vs. low-carb study where researchers
educate groups of volunteers and let
them eat self-directed low-fat or lowcarb diets in their own homes to see
how they fare.
14

One important concept to understand before reading

as their own control group. Metabolic ward studies

through this breakdown is that the study was not look-

are where trial participants are strictly monitored to

ing at the real-world efficacy of diet interventions. That

measure energy expenditure and energy intake. These

is to say, this was not a free living low-fat vs. low-carb

ward studies are considered the gold standard in diet

study where researchers educate groups of volunteers

trials as free-living studies often rely on far less accu-

and let them eat self-directed low-fat or low-carb diets

rate self-reported data. Patients included were required

in their own homes to see how they fare. The inves-

to have been weight stable for the past 6 months and

tigators designed this intervention to examine some

were screened to ensure they were otherwise healthy

specific mechanisms of weight loss discussed in the

(i.e. free from diabetes, chronic illness, eating disorders,

sections below.

etc ). The purpose of the trial was to determine if a

reduction of carbohydrates in the diet would confer a fat

One version of the insulin hypothesis states that in

order to lose body fat you must restrict carbs to bring
down insulin, high levels of which will prevent fat
loss. Dr. Halls study has been designed to test this
hypothesis to see if reduced-carb diets confer a fat
loss advantage over reduced-fat diets when calorie
intake is strictly controlled.

loss advantage above and beyond a reduction in energy

intake. To test this hypothesis, Dr. Halls research team
reduced equal caloric amounts of carbs and fats in the
restricted fat and restricted carb groups to determine
its effects on energy expenditure, nutrient oxidation, fat
loss, and bodyweight. The reduced-carb group saw a
30% caloric reduction from carbs alone and the reducedfat group saw a 30% caloric reduction from fat alone.

Who and what was studied?

Subjects underwent 5 days eating a baseline diet fol-

Nine women and ten men with obesity were recruited

diets. The macro balances of each diet are shown in

for this randomized, controlled, cross-over metabolic

ward study. A cross-over trial is when all patients receive
both treatments at different periods, essentially acting

lowed by 6 days eating one of the two calorie-restricted

Figure 1. The baseline diet was 2,740 calories (50%
carbohydrate, 35% fat, 15% protein) and the restricted
calorie diets were both 1,918 calories. The restricted fat

Figure 1: Macronutrient contents of the diets as a percent of total calories

15

group cut out 828 calories of fat and the restricted carb
group cut out 840 calories of carbohydrates. Protein
intake was kept constant throughout. Of particular
note was that sugar consumption did not decrease in
the reduced-fat group compared to baseline. In fact, it
went up from 152 grams/day to 170 grams/day. It was
important to keep sugar intake up as to not cause any
decreases in insulin secretion, which would have confounded the study results.
All the volunteers were crossed-over from one diet to
the other, as they went through a 2 to 4-week washout
period between the restricted fat and restricted carb
diets. Food intake was meticulously monitored. All
subjects were confined to the metabolic ward for the
entirety of the study and were made aware of how critical it was to consume all food provided to them. Even
when subjects were visiting with friends and family,
they sat in a common area under the observation of
research staff to ensure no food was being passed off.
Daily exercise was also required. Sixty minutes of treadmill walking at a self-selected fixed pace was required
everyday that patients were in the metabolic ward.

It was
important to
keep sugar
intake up as
to not cause
any decreases
in insulin
secretion, which
would have
confounded the
study results.

Multiple measurements were taken over the course

of this trial including cholesterol, appetite hormones,
insulin, cortisol, and body fat percentage. Though a
dual-energy X-ray absorptiometry (DXA) scanner
was employed to assess body fat, this method is not
sensitive enough to pick up the small changes in body
fat loss that occurred over the duration of this trial.
To get a more sensitive measurement, the changes in
body fatness were determined using net fat balance
by indirect calorimetry while residing in a metabolic
chamber, in combination with measures of nitrogen
loss in urine. Essentially the difference between dietary
fat intake and net fat oxidation (fat oxidation minus
de novo lipogenesis) were used to measure overall fat
mass loss. Although this method cannot tell us where
the fat is being lost from, a sensible prediction would be
that most would come from adipose tissue. However, it

is possible that some fat could be lost from the liver or

muscles, which would also be beneficial.
A mathematical model of human metabolism was
employed to predict trial outcomes and to help extrapolate the 6-day results. Data from the participants results
were plugged into this model to predict how they would
continue to lose weight over the course of 6 months. Dr.
Halls model has undergone some extensive validation
and has been shown to be a fairly accurate predictor of
weight gain and loss in adults 18 years of age and older.
His research at the National Institute of Health has been
used to create the Body Weight Planner, which you can
explore on their website. A brief instructional video can
be found here.

16

burned carbs as their main fuel source throughout the

19 healthy subjects with obesity were randomized

trial and saw little reduction in fat oxidation. An inter-

and crossed-over into both a restricted fat and

esting caveat that popped up was that protein oxidation

restricted carb diet under strict observation in a met-

was increased in the reduced carbohydrate group, indi-

abolic ward. Indirect calorimetry was used to assess

cating that the higher carb intake of the reduced-fat

fat mass loss over both 6-day periods participants

group may have a slight protein sparing effect. Some

were on restricted diets, and a mathematical model

may worry that this increased protein oxidation equates

was used to predict how much weight would be lost

to muscle tissue being broken down. However, this

over 6 months.

may not necessarily be the case as the protein oxidation

could be coming from the amino acids in the diet. As

What were the findings?

The results are summarized in Figure 2. As may have
been expected, the reduced-carb group shifted to primarily oxidizing fat as fuel, and reached a steady state
after about 4 days. The reduced-fat group consistently

most reduced-carbohydrate diets are typically paired

with an increased protein intake, it is unlikely that any
muscle wasting would occur.
One interesting finding was that the reduced-fat group
did not experience a major shift in fat or carb oxida-

Figure 2: Summary of the study and results

Adapted from: Hall, KD et al. Cell Metab. 2015 Sep.

17

tion the way the reduced-carb group did. Within the

reduced-carb group, fat oxidation went up 403 calo-

How Glycogen Affects

Weight Loss

ries (~45g) per day and carb oxidation went down 520
calories (~130g). This shift to primarily utilizing fat as
energy is a known effect of low-carb diets. One might
speculate that a high carb diet would see an equally
dramatic shift towards burning carbs as the primary fuel, but the reduced-fat group saw fat oxidation
decreased by only 31 calories (~3.4g) per day and carb
oxidation increase by 44 calories (~11g). It seems that
when faced with a large reduction in dietary fat intake
the body keeps trucking along, burning fat and carbs at
approximately the same levels.
Overall, the reduced-fat diet lead to a fat mass loss of
~463 g and the reduced-carb diet saw a fat reduction of
~245 g. The difference in these numbers can possibly be
explained by the stored glycogen the reduced-carb group
would have burned off in the first 2 to 4 days of the 6-day
diet period, after which the fat mass loss would more
closely match that of the reduced-fat group. The fat loss
seen in the reduced-fat group occurred even though no
significant changes in 24-hour insulin secretion were
seen. By contrast, the reduced-carb group saw a 22%
reduction in 24-hour insulin secretion. This finding
clearly demonstrates that a reduction in dietary carbohy-

Within this 6-day trial we saw the reducedfat group lose more fat mass than the
reduced-carbohydrate group. But this is not
necessarily because the fat restricted diet
provides a significant fat burning metabolic
advantage. The most likely explanation for
why restricted-fat came out on top was that
the reduced carb group was burning through
their glycogen stores in the first few days of
the trial.
The human body can hold about 2,000 calories worth of glycogen in the skeletal muscles
and liver. When the reduced-carb participants
were switched from their baseline diet of
350g carbohydrate down to 140g, they began
to use up their glycogen stores as their bodies
started to adapt to preferentially burning fat.
Because the body was utilizing these glycogen calories it was not using fat calories. Once
the glycogen stores had been depleted by
about day 4, the reduced-carb dieters then
reached a steady-state of fat burning.

drate and insulin is not necessary for losing fat mass.

Figure 3 depicts the results from when the subjects
data was plugged into the human metabolism model. It
predicted that the reduced-fat diet would see about 3
kg (6.6 lbs.) greater fat loss after 6 months, a 40% difference in fat loss. Of course, this was assuming that
participants would adhere 100% to the diet. Real world

Even though the mathematical model predicted an advantage to reduced-fat dieting

in the long run, the utilization of these glycogen stores by the reduced-carb group are
likely a significant contributor to why the carb
restricted diet only experienced about half
the fat loss in this 6-day window.

diet studies tend to show us that compliance starts to

dwindle after about the 6-month mark. Additional
simulations were run to see what would happen if carbs
were dropped even lower in the reduced-carb group
with subtracted carb calories being swapped out for fat
to keep total calories constant. The model predicted
that the very low-carbohydrate diet (<50g/day) would

experience comparable weight loss to the very low-fat

diet, minimizing the 3 kg (6.6 lbs.) difference seen in
the original prediction.
Small caveats also included the significant reduction in
sleeping metabolic rate and total energy expenditure
18

Figure 3: Mathematical modelling prediction of diets 6 months out

seen in the reduced-carb diet that was not seen in the

out (CICO). A common interpretation of CICO is that

reduced-fat diet. This is suggestive of some more subtle

there should be few if any differences between diets of

metabolic changes that occur due to the effect of certain

equal calories on fat loss or energy expenditure. This

dietary macronutrient compositions.

study shows us that while that strict interpretation of

CICO is not 100% correct, it is pretty darn close. While

The reduced-fat diet group lost more fat mass than

then reduced carbohydrate group. The reduced-fat
group did not see any significant decreases in insulin production, demonstrating that reducing insulin
levels is not necessary for losing fat mass. The mathematical model of human metabolism predicted an
advantage to the low-fat diet over the course of 6
months, but the differences were minimal and all but
disappeared when a very low-fat diet was modeled
against a very low-carb diet.

the CICO model holds approximately correct over most

of the macro spectrum, the mathematical model predicted that it does start to break down a little bit when
looking at macronutrient extremes. As we saw in Dr.
Halls 6-month model prediction, the reduced-fat group
had a slight advantage over the reduced-carb group.
These small differences are about the extent to which
you may see any difference between diets. And as noted
earlier, that advantage all but disappeared when very
low-carb was compared to very low-fat diets.
While the study was incredibly rigorous in its design

What does the study really

tell us?
This study lends more credence to the theory of energy balance, otherwise known as calories in, calories

and execution, the sample size was small. Only 17 of

the 19 recruited individuals completed the entire study.
By metabolic ward study standards, 17 is actually a
pretty large sample size and provided and enough participants to ensure small differences in fat loss could
19

be detected. However, because of the small sample

That isnt to suggest that low-carb diets should not be

size it may be difficult to extrapolate these results to

employed if that is your preference. The higher protein

the general population. One should also note that the

intake that is often paired with low-carb diets can help

participants in this study were relatively healthy, so the

to increase satiety, causing you to feel less hungry. Many

results here may not extend to people with health issues.

may find a reduced-carb diet easier to adhere to than

People with other health issues may also be on various

a reduced-fat diet. People who are insulin resistant, a

medications that could alter metabolism, but such peo-

condition commonly found among those with pre-di-

ple were excluded from this study. These factors make

abetes or type 2 diabetes, can often experience better

any generalizations from this study to such populations

very challenging.

While a calorie might not be exactly a calorie, it is

pretty close in terms of its effects on metabolism
during periods of weight loss. Small shifts can occur
depending on the macronutrient composition, but
the end results on equally caloric low-carb and lowfat diets are not strikingly different. Due to the small
sample size and the type of patients recruited to this
study, extrapolation of the results is limited.

The big picture

The practical implications we can take away from
this study are very limited, but we can surmise that a
reduction in insulin secretion brought about through
low-carb dieting does not seem to confer any metabolic
advantage for fat loss. In a way, this is both good and
bad news. The bad news is that a low-carbohydrate diet
does not appear to possess any super fat-blasting properties which, had that been proven true, would have
been great news to dieters everywhere. If this paper
had shown a significant advantage to low-carb dieting
it very likely would have been a game changer in how
we approach the treatment of obesity and weight loss
research. The good news is that, because a low-carb is
not necessary for fat loss, more eating styles are available
to those trying to lose weight. If you are not someone
who likes low-carb dieting, higher or moderate carbs
diets are a perfectly viable option for weight loss.

If this paper
had shown
a significant
advantage to
low-carb dieting
it very likely
would have
been a game
changer in how
we approach
the treatment
of obesity and
weight loss
research.
20

results on a low-carbohydrate eating plan. In a real-

the bodys energy systems actually make the transi-

world setting, adherence is king. Even if low-carb diets

tion from preferentially burning carbs to preferentially

had the ability to melt fat off your body, if you are not

burning fat rather quickly. Within the reduced-carb

able to stick with the diet it will not be an asset for your

group of this study, it took about 4 days before they had

long-term weight loss goals.

reached maximum fat oxidation and we began to see a

leveling off. This observation is corroborated by other

The fact that low-carb diets do not confer a superhuman ability to lose fat mass is a little disappointing. A
diet that did possess such properties would be a most
welcome finding. However, this study does reinforce
the fact that most any diet, be it Mediterranean,
DASH, paleo, or vegetarian, can all work quite effectively for weight loss. It all comes down to personal
preference and the ability to stick with the diet in the
long term.

trials that show the same quick fuel transition. Once the
fat oxidation plateau has been achieved, it remains very
constant over the following weeks. Hence, 6 days would
have been sufficient time for subjects to achieve maximum fat oxidation on the reduced-carb diet.
On the criticism that the reduced-carb diet was not
ketogenic, they are correct if you are defining ketogenic
as 50 grams of carbs a day or fewer. But if the argument
is that being in a ketogenic state confers bonus fat burning abilities, youd think there might be at least some

Frequently asked questions

XXL edition
The present study was full of intricacies and nuances.
In other words, it was ripe for misinterpretation by the
popular media. You may have seen flashy headlines
declaring the superiority of low-fat diets and lamenting
the death of low-carb. With so much hyperbole surrounding this study in the news and blogosphere, were
bringing you an XXL edition of the F.A.Q. in order to
bust some common myths, misconceptions, and criticisms surrounding this trial.

A 6-day study is not long enough to get into

ketosis or to become fat adapted.
A lot of people have been commenting on the short
duration of this study. Many argue that it takes up to
1 month to become properly fat-adapted or that the
carb content in the reduced-carb diet was not low-carb
enough to induce a state of ketosis. This misconception
about fat-adaptation likely stems from those who have
gone low-carb and felt hazy or foggy, commonly known
as the low-carb flu, for 2 to 4 weeks. While it may
take some time to feel normal again on a low-carb diet,

suggestion of a dose-response curve as carbs in the diet

decrease. This means we should be able to see fat loss
increase as carbs in the diet decreased. No such dose
response was observed in this trial. The mathematical
model employed also indicated that a very low-carb diet
would have similar fat loss results to a very low-fat diet.
Currently, no metabolic ward study of a ketogenic vs
non-ketogenic diet exists, where calories and protein
are held constant. However, there have been non-metabolic ward studies indicating no metabolic advantage
to ketogenic diets. Dr. Hall has just completed (but not
yet published) an 8-week metabolic ward study that will
hopefully shed some more light into this area of research.

Nothing can be gained from this study because

it does not represent real world conditions.
This study was not about which diet leads to better
results under real-world conditions. There are many
other studies out there that have attempted to address
that question, but as mentioned before, a successful
diet comes down to adherence. The authors were very
upfront in what this trial was designed to study and its
real-world applications. The research team planned this
21

study to look at specific mechanisms of fat loss, primar-

not allowed to participate. While this may seem odd

ily testing if a reduction in insulin is necessary to lose

at first, it was implemented because neuroimaging

body fat.

was used on all participants, most likely to be used in

future publications. Those who are right-handed tend

Dr. Hall does have a study in the works that will be

to perform tasks in either the right or left side of the

looking into some more real-world diet issues. His

brain, whereas left-handers tend to split that task evenly

future trial will be examining some of the changes in

across the brain. If you are using neuroimaging to look

metabolism and the brain that may lead to weight loss,

at a specific part of the brain, this difference in brain

plateau, and regain.

hemisphere usage in right and left handers can throw

off your results.

The authors even stated the following in their discussion:

Translation of our results to real-world weight-loss

What about the hiccups in the study where people receive

diets for treatment of obesity is limited. We did

incorrect meals and one womans data was not included?

not address whether it would be easier to adhere to

In any clinical trial, mistakes are bound to happen. In

a reduced-fat or a reduced-carbohydrate diet under

this case, one male and one female participant received

free-living conditions. Since diet adherence is likely

the wrong meals on the first day the reduced-carb and

the most important determinant of body fat loss, we

reduced-fat diets were administered. The researchers

suspect that previously observed differences in weight

opted to keep these data in the final analysis, as remov-

loss and body fat change during outpatient diet inter-

ing them did not affect the statistical significance of any

ventions were primarily due to differences in overall

comparisons.

calorie intake rather than any metabolic advantage of

a low-carbohydrate diet.

Two of the male participants also dropped out of the

study after finishing the reduced-carb phase of the trial.

Why were obese but metabolically healthy people

Their data for the reduced-carb portion was kept in,

selected? Wouldnt having obese people who were meta-

but obviously they did not contribute any data to the

bolically unhealthy have made more sense?

reduced-fat phase.

It is possible that a future study like this may be performed in those with obesity and metabolic syndrome,

Curiously, one female subject saw some unusual

but the additional factors that come with metabolic

measurements on her DXA scans that prompted the

dysfunction complicate the results of the study. For

research team to exclude her data from that partic-

example, someone with type 2 diabetes operates under

ular analysis. This was because the DXA showed her

a different metabolic paradigm than someone without it

fat mass had increased on both the reduced-carb and

due to insulin resistance and potentially decreased pan-

reduced-fat diets despite the fact that she had experi-

creatic function. Furthermore, many type 2 diabetics

enced weight loss and was in negative energy balance.

may be taking medications that alter their metabolism

Gaining fat mass while in substantial negative energy

which adds more confounding variables to the mix

balance is something that is physiologically impossible,

when trying to draw conclusions.

making it a clear outlier and hence leading to the decision to exclude those data points.

Why were left-handed people excluded from the trial?

If you look at the exclusion criteria for this study, you

Why did they compare a low-fat to a moderate-carb

will indeed find that those who were left-handed were

diet instead of a low-fat to low-carb diet?

22

The baseline diet was set at 35% fat, 15% protein and

going on in Table 4? Try not to read too heavily into the

50% carbohydrate and about 20% of those total calories

blood data presented, as they were all exploratory sec-

were from sugar. This is believed to represent a typical

ondary endpoints of the study. The p-values were also

American diet composition. Because of this compo-

uncorrected for multiple comparisons.

sition, it was impossible to make subtractions from

carbs any lower in the low-carb group without having

Can we see the individual data?

to add fat calories back in while keeping total calories

Dr. Hall has said that he will be publishing future papers

constant between groups. The researchers did not want

exploring the correlates of individual responses seen in

to do this, as the whole point of the trial design was to

this study. We look forward to seeing these data too!

change just one macronutrient level while leaving the

others untouched. This is why the macro composition

What if this restarts the low-fat diet trends again? I

was askew after the pre-set number of calories and been

loved the 80s!

subtracted from each group.

Please, no more high vs low-fat diet shenanigans! Both

dietary approaches are perfectly healthy. Pick what

There were too few participants in the study.

works best for you in the context of your food prefer-

Usually, before a trial is conducted, a power calculation

ences, environment, and health status.

is used to determine how many people may be needed

in the trial to reach adequate statistical power. That is
to say, how many people will be needed to ensure that
a statistically significant difference can be found in a
study when there is one in reality. This method is how
Dr. Hall reached the number 19 for participants needed
in this study. It should be noted that due to the complexities and costs of running a metabolic ward study,
19 is actually a large sample size, comparatively.
Why did the reduced-fat group experience a greater
drop in fasting blood glucose compared to the reducedcarb group (and other oddities in Table 4)?
You would expect the reduced-carb group to have the
greater decrease in endpoints such as fasting blood glu-

What should I know?

The most direct takeaway from this study is that carbohydrate restriction and insulin decreases are not
required for fat loss. For a more real-world implication,
we can extrapolate that you should pick whichever diet
you can adhere to in the long run. This study is not
showing that low-carb diets are ineffective, but rather
demonstrates that both a low-carb and low-fat option
may be equally efficacious for those seeking fat loss (at
least as far as your body is concerned). Decreased insulin
in otherwise healthy subjects will not provide an additional fat loss advantage, so do not fret that you must go
low-carb or you will never lose weight ever again.

cose and fasting insulin levels. In this case that did not
happen, as the reduced-fat group dropped their fasting

The king of all discussion topics: low-carb dieting. To

glucose by 7.1 mg/dl and the reduced-carb group only

discuss the topic (actual discussion, not heated opin-

experienced a 2.69 mg/dl drop. Decreases in fasting

ions!), check out the private ERD Facebook forum.

insulin were comparable between groups. So whats

23

Investigating
vitamin D as a
performance
enhancer

A Systems-Based Investigation
into Vitamin D and Skeletal
Muscle Repair, Regeneration and
Hypertrophy

24

Introduction

inadequacies may be a decrease in the bodys ability to

Why is Vitamin D such a popular supplement, among

both researchers and the general public? One reason is
that it potentially acts on a broad spectrum of health
and performance measurements. Researchers have
examined the effects of this fat-soluble vitamin on
chronic pain, cystic fibrosis, multiple sclerosis, and the
risk of cardiovascular disease and mortality (covered
in ERD #7).

regenerate muscle tissue. Researchers have known since

1985 that there are vitamin D3 receptors on muscle cells,
and in the past few years research on vitamin D in the
context of boosting performance has become popular.
One intervention trial in well-trained athletes showed
an improvement in sprint times and vertical jump with
supplementation. Further studies have associated higher
vitamin D status with a more rapid recovery of skeletal
muscle strength after an acute bout of intense exercise.

Many people are not getting enough of this vitamin.

Recent estimates have indicated that 37.3% of the
worlds population may have an inadequate level of
vitamin D. The Institute of Medicine has defined blood
concentrations between 30 to 50 nmol/L of vitamin
D to generally be considered inadequate. Although
sometimes defined at different cutoff points, risk of
deficiency can be characterized by levels lower than
30 nmol/L, while an adequate, healthy range is usually
considered to be between 50 and 125 nmol/L.
Well be using units of nmol/L throughout this article,
although many labs report vitamin D levels in ng/mL.
You can see how these two units relate to each other
in Figure 1. One possible downstream effect of these

The growing body of data showing that vitamin D plays

an important part in the function of skeletal muscle
suggests this vitamin may be a potential ergogenic
aid. The fact that vitamin D3 is a relatively inexpensive and widely-available supplement makes it all the
more attractive to athletes. Around 56% of athletes
have vitamin D levels of 80 nmol/L or lower, so supplementation could help combat deficiency. Although
previous research has indicated vitamin D may help
with improved muscle healing, a causal relationship
has not yet been firmly established. The study under
review looked further into this connection to determine if there is a potential cause-effect relationship
between vitamin D and muscle repair, regeneration,
and hypertrophy.

Figure 1: Converting nmol/L to ng/mL

25

that respond to the supplement. By using a combined in

Vitamin D plays diverse roles in maintaining health,

vivo/in vitro design, the researchers were able to exam-

and has been investigated for beneficial effects on a

ine the impact vitamin D had on muscle recovery in

host of conditions. A developing line of research has

humans while also attempting to determine the cellu-

looked into the roles this vitamin could play in the

lar mechanisms through which the effect might occur.

context of boosting physical performance. The pres-

Essentially, the researchers were trying to figure out

ent study investigated vitamin D to determine how it

how vitamin D affects muscle repair and how it does

affects muscle repair, regeneration, and hypertrophy.

what it does.

Who and what was studied?

First, the researchers conducted a double blind, randomized controlled trial in humans (in vivo) to
investigate the effects of vitamin D on the muscles
ability to recover from exercise-induced damage. The

Researchers recruited twenty young, physically active,

and healthy males for the in vivo study. Baseline
vitamin D status was assessed and participants were
excluded if they had adequate vitamin D concentrations, defined in this study as greater than 75 nmol/L.
All vitamin D blood measurements were analyzed

overall study design is shown in Figure 2.

using an analytical chemistry technique considered the

Second, they extracted isolated muscle cells (in vitro)

MS). The average vitamin D status of the cohort was 45

gold standard for assessing vitamin D levels (LC-MS/

from humans that were vitamin D insufficient and

exposed them to 1,25-dihydroxyvitamin D (1,25(OH)
D), the biologically active form of vitamin D. This was

nmol/L. Participants were randomized into the control

or intervention groups and received six weeks of either
supplemental vitamin D3 (4,000 IU/day) or placebo.

done to identify the aspects of muscle cell regeneration

Figure 2: Trial design

26

Whats the difference between vitamin D2 and D3 ?

Vitamin D in the diet comes in two forms: D2 and D3. D2 is usually found in fungi, while D3 is
found in animal products.
D2 does not bind well to the human vitamin D receptor, compared to D3. For that reason,
and because it is more easily deactivated and thus remains in an active form for less time,
it is generally considered to be a poorer form of the supplement.
An overview of vitamin D metabolism is shown in Figure 3. Vitamin D2 and D3 must both
undergo further enzymatic reactions before the body can utilize them. They both travel
to the liver, where they are converted into 25-hydroxyvitamin D (25(OH)D), the form that is
usually measured in blood tests for vitamin D. Vitamin D3 is more effective at raising this
measurement than D2 is. They are then sent to the kidneys, where the final conversion
step occurs. 25(OH)D becomes 1,25-dihydroxyvitamin D (1,25(OH)D), the bioactive hormonal form of the vitamin.
It is this substance that can interact with the vitamin D receptors found throughout tissues
in the body. The 1,25(OH)D that is not used by the body gets degraded into inactive forms,
with D2 tending to be deactivated more quickly than D3 .

Figure 3: Vitamin D metabolism

Adapted from: Dahlquist et al. J Int Soc Sports Nutr. 2015 Aug.

27

Before the participants began their supplement regimen,

baseline muscle recovery measurements were taken.

This study was split into two parts: one performed on

Participants produced a maximal voluntary contrac-

humans and one on cultured muscle cells. During the

tion (MVC) in their right leg prior to and following a

human study, researchers administered exercise tests

series of eccentric exercises. An eccentric contraction

before and after a six-week intervention during which

is the portion of the exercise during which a muscle

participants either took vitamin D3 or a placebo. The

is lengthening. For example, the eccentric portion of

researchers were attempting to determine if vitamin D

a bicep curl is when the weight is lowered back down

plays a role in muscle repair. During the second study,

to the rest at the side, thus lengthening the biceps. The

the cultured muscle cells were given varying doses

eccentric exercises and MVCs were performed on

of vitamin D3 to try and determine the mechanisms

an isokinetic dynamometer, which can measure the

through which the vitamin acts on muscle tissue.

amount of torque being produced. A video of this

machine in action can be viewed here. MVC torque was
then re-measured 24 hours, 48 hours and seven days
following the exercise session. This same procedure was
repeated following the six weeks of supplementation.
For the in vitro study, researchers took muscle biopsies
from the quadriceps of fourteen young male volunteers with inadequate levels of vitamin D. The muscle
cells were cultured and isolated before the experiment
began. To induce muscle damage, a pipette was used to
scrape the cells on the culture plate. Once any remaining cell debris had been removed, the muscles cells were
exposed to a low (10 nmol/L) or high (100 nmol/L) dose
of vitamin D3, or a control substance. These varying
doses were used to determine if the potential responses in muscle repair are dose dependent. Researchers
observed the cells at zero and 48 hours, as well as seven
and 10 days after the pipette-induced cell damage.

What were the findings?

The first, and perhaps most obvious finding, was that
there was a significant increase in serum vitamin D in
the group receiving the supplement. Concentrations
increased by a factor of 2.5, from an inadequate vitamin
D status of 45 nmol/L to an adequate status of 115 nmol/L. On the other hand, the placebo groups serum level
actually dropped by 26%, from 45 nmol/L to 33 nmol/L.
Two exercises were utilized to test muscle recovery. One
where participants produced maximal torque while
moving their legs at 60 degrees per second and one at
180 degrees per second. Within the vitamin D group,
significant muscle recovery improvements were seen in
the 60/second test and, although a positive trend was
observed, no statistically significant results were record-

The first, and perhaps most obvious

finding, was that there was a significant
increase in serum vitamin D in the
group receiving the supplement.
28

of the participants to recover their strength after eccen-

What does the study really

tell us?

tric exercises (i.e. peak torque) was highly correlated (r2

This study illustrates the important role that vitamin

= 0.88). No changes in muscle soreness were detected

D plays in muscle recovery, repair, and regeneration.

between groups.

When serum vitamin D status is brought up from a

ed for the 180/second test. However, the relationship

between higher serum vitamin D levels and the ability

level of inadequacy, the functional recovery of skeletal

The in vitro study looked at endpoints like cell migration,

muscle occurs more quickly. The cell cultures tested in

which is a key initial step in the muscle repair process.

this study provide a deeper understanding of how this

Both the high and low doses significantly enhanced cell

process occurs, namely by illuminating the underlying

migration compared to placebo, but the high dose was

mechanisms that heal muscle tissue.

superior. The high and low doses also improved migration speed and distance covered, which resulted in more

Data presented in the study may also provide insight

cells moving into the wounded space to repair the dam-

into what might occur if supplemental vitamin D is

aged tissue. Interestingly, the high dose of vitamin D3

taken in excess. When tested on the muscle cells, the

did not perform as well as the low dose when it came

higher vitamin D dosage suppressed certain aspects

to myoblast fusion. Myoblasts are cells that fuse to form

of the muscle repair process. Myoblast fusion, creatine

myotubes. Myotubes are used to produce various mus-

kinase activity, and certain gene expressions were not

cle groups required to generate force.

as active as their low dose counterparts by day 10 of the

cell culture observations. It is unclear if these same sup-

The low treatment group experienced significant

pressions would happen in vivo and, if so, what dose

improvement in myotube measurements over the con-

would be needed to bring about these effects.

trol and high vitamin D3 treatments. Cells exposed to

the lower dose also saw increased creatine kinase activity,

While the dosage used in this study (4,000 IU/day)

which helps muscles to function effectively by energizing

demonstrated a positive effect on muscle repair in

creatine. Low doses also showed a trend in being able

people with insufficient levels, it is not known if these

to up-regulate certain gene expressions associated with

effects would continue to improve, level off, or dissipate

muscle repair and growth, more so than the high dose.

at higher doses. The safe upper limit established in the

United States and Canada is presently set at 4,000 IU/

During the human intervention portion of the trial,

higher vitamin D status was positively correlated with
the ability of muscles to recover quicker from exercise-induced damage, although statistically significant
differences were only seen in one of the two muscle
recovery tests. When looking at the results of the
muscle cell cultures, the low dose of D3 appeared to be
the most beneficial for muscle fiber repair, improved
creatine kinase activity, and up-regulation of gene
expressions involved with muscle tissue growth.

day, but some research suggests that the upper limit

could be as high as 10,000 IU/day. However, a dosage of
1,000-2,000 IU/day should be sufficient for most people.
The study did have a few design limitations. A sample
size of 20 participants is fairly small, so a larger scale trial
is needed to help verify the results of this study. Further
studies should include female participants as well. Also,
the chosen mode of exercise was highly specific and
is not necessarily translatable to everyday activities or
training. Future experiments could employ exercise
modalities that reflect more real-world scenarios.
29

Furthermore, the doses of D3 the muscle cell cultures

mone properties has led scientists to investigate vitamin

were exposed to was well above what would be seen in

D as a potential ergogenic aid. Specifically, the potential

a normal human, making the different doses used in

ability of vitamin D to assist in rapid muscle recovery,

the in vitro and in vivo studies hard to compare. The

which would be of particular interest to athletes.

authors suggest that further research should look into

estimating the concentration of D3 that skeletal muscles

The authors point out that this research has raised new

are normally exposed to. These discoveries would help

questions about populations susceptible to muscle

researchers design in vitro experiments that are more

damage and vitamin D inadequacy, such as the elderly.

relevant to real-world concentrations of vitamin D.

A more senior population may experience a diminished

capacity to regenerate muscle when vitamin D levels

People with inadequate serum vitamin D can increase

intake to ensure adequate levels, which will likely
benefit muscle repair and regeneration. In vitro studies demonstrate that vitamin D is able to enhance
these repair mechanisms. Future testing is needed to
determine if doses higher than 4,000 IU/day will benefit or potentially attenuate these mechanisms.

are not sufficient. Higher levels of supplementation may

aid in reversing some of these processes.
Previous in vitro studies conducted on human cells
have shown promise in the use of vitamin D to aid in
muscle recovery times. An animal study, performed on
rats, echoed these findings and demonstrated enhanced
recovery with the supplement. Besides the current
study, one additional study has investigated vitamin Ds
muscle recovery effects in humans. Baker et al. exam-

The big picture

Views on vitamin D have changed in the past decade. It
was previously mostly viewed as a regulator of calcium
balance, hence aiding in healthy bone formation. For
example, rickets, a bone disorder typically found in
children, is caused by insufficient vitamin D intake and
can be reversed with supplemental vitamin D. However,
research performed over the past decade has shown that
many tissues in the body
will respond to and function sub-optimally when
exposure to vitamin D is
limited. Studies have identified over 900 genes that
are regulated in part by
vitamin D. The biologically
active form of vitamin D
shares some characteristics
with steroids like testosterone. Thus, research
looking into these hor-

ined 28 healthy active males with adequate vitamin

D levels. After baseline measurements were recorded,
4,000 IU daily of vitamin D or placebo was administered for 35 days. The post-test results showed both
enhanced recovery time and a decrease in biomarkers
of muscle damage in the vitamin D group.
The combined results of the muscle cell tests, animal

Studies have
identified over
900 genes that are
regulated in part
by vitamin D.

studies, and experiments

performed in humans
have built a strong case
for the need to ensure
adequate vitamin D status
for optimal physical performance. However, the
research looking into supplementation in females
is lacking, as both of the
two human intervention
trials recruited only fit
healthy males.
30

The last option is to harness the internal production of

While typically viewed as the vitamin that aids in

vitamin D that occurs after sun exposure. When the

bone formation, vitamin Ds role in the body has

skin is exposed to ultraviolet B radiation (UVB), it sets

been shown to be wide ranging. An emerging body

into motion a process that produces biologically active

of evidence has demonstrated that inadequate levels

1,25(OH)D, the form of vitamin D thats useable to our

of vitamin D can lead to decreases in muscular per-

cells. Going outside for five to twenty minutes, two to

formance and increased recovery times. Athletes and

three times a week, with at least 5% of skin exposed can

the elderly in particular may want to monitor their

help generate adequate amounts of D3. Fifteen min-

vitamin D status. Future trials should look to inves-

utes of UVB exposure during the summer in bathing

tigate these effects in females, as the present research

suit attire (before sunscreen application) can produce

has been conducted solely in healthy, fit males.

10,000 to 20,000 IU of D3 in light-skinned individuals,

while those with darker skin need much longer exposure times. However, as UV radiation from the sun or

Frequently asked questions

tanning beds is a known carcinogen that plays a role in

get my vitamin D?

very fair skin), limiting exposure of unprotected skin

Supplementation vs. Food vs. Sunlight: How should I

There are many options to ensure adequate intake of

vitamin D. Vitamin D comes in two flavors: D3, mostly
found in animals, and D2, the form typically seen in
non-animal sources. Certain high vitamin D foods can
be used to help defend against vitamin deficiency. Fish
like halibut, salmon, trout, mackerel, and sturgeon are
abundant sources of vitamin D3. Other good options
may include eggs, certain fortified cereals, and fortified
dairy products. Sources of D2 include many varieties of
mushrooms, such as portabella and shiitake, and fortified soymilk.
Sometimes food may not be enough. In these cases, a
moderate supplemental dose of 1,000 to 2,000 IU/day
of D3 may suffice for most people, although at-risk populations such as those who cover their skin extensively
or who have very dark pigmentation could warrant
higher doses. Studies have shown 100 IU of additional
vitamin D per day can be expected to increase levels
by about 2.5 nmol/L on average. So, a person with a
starting vitamin D level of 37 nmol/L would need about
1,500 IU/day to bring levels up to 75 nmol/L. Luckily,
D3 tends to be an inexpensive supplement.

the development of skin cancer (especially in those with

to sunlight would be prudent for some people. Many
factors can affect these sunlight-driven synthesis rates
though, such as higher altitudes, cloudy climate, thick
ozone layers, and skin pigmentation.

What should I know?

The results of this trial demonstrate that low levels of
vitamin D can easily be elevated to a healthy range via
supplementation, and that this may benefit muscle
repair and recovery.
The next step in progressing this line of research would
be to try and replicate these results using a larger sample
size, while ideally including female participants. If you
believe you may have low vitamin D, get your 25(OH)D
levels assessed by your doctor before adding a D3 supplement, as it can interact with some medications.

Oh Vitamin D, what cant you do? The answer: quite a

lot actually although this particular study was promising for a subset of the population. To discuss the rich
area in between panaceas and snake oil, head over the
to the ERD Facebook forum.

31

Not-so-safe
supplements

Emergency Department Visits

for Adverse Events Related to
Dietary Supplements

32

Introduction
Dietary supplements are sometimes erroneously perceived as inherently healthy. And because of the way
many supplements are advertised, its easy to overlook that
improper administration can lead to adverse outcomes.
The classification of a supplement is defined in
the United States Dietary Supplement Health and
Education Act of 1994 (DSHEA) as a vitamin, mineral, herb or botanical, amino acid, and any concentrate,
metabolite, constituent, or extract of these substances.
In the U.S., the Food and Drug Administration (FDA)
is the governing body that oversees the regulation of
dietary supplements. If a supplement has been reported to be causing serious adverse events or reactions,
the FDA has the authority to pull it from the market.
However, no safety testing or FDA approval is required
before a company can market their supplement. The

ments. An independent survey has echoed these results,

finding that 67.2% of respondents felt extremely or
somewhat confident in supplement efficacy and 70.8%
felt extremely or somewhat confident about their safety.
While the majority of Americans trust in their supplements, more than one-third have not told their
physician about using them. There are numerous documented drug-supplement interactions ranging from the
mild to the severe. The herb St. Johns Wort is thought
to be able to reduce symptoms in people with mild to
moderate depression. But this natural supplement also
has 200 documented major drug interactions, including
some with common depression medication. However,
no good data currently exists to document how common adverse events related to dietary supplements may
be. The authors of the present study have used surveillance data to try and fill this knowledge gap.

lack of oversight authority given to the FDA has even

drawn the attention of late night talk shows hosts like

Due to DSHEA, supplements remain largely unreg-

John Oliver, who humorously covered the issue in this

ulated by the FDA. But dietary supplements are

YouTube video.

becoming ever more popular, as about half of U.S.

adults report using one or more in the past 30 days.

Many adults are using one or more supplements to

Trust in the safety and efficacy of these supplements

address illnesses or symptoms, and to maintain or

also remains high. The authors of this study aimed

improve health. Half of all U.S. adults have report-

to investigate how many annual adverse events are

ed using at least one supplement in the past 30 days.

caused by improper supplement usage.

Twelve percent of college students have reported taking

five or more supplements a week. Now, more than ever,
there are seemingly endless options to choose from.
The number of supplement products currently available on the market is thought to be in excess of 55,000.
Compare that to the mere 4,000 available in 1994, when
DSHEA was passed.
Furthermore, confidence in the safety and efficacy of
these supplements is very high despite the lack of rigorous oversight by the FDA. A survey conducted by the
trade association, Council for Responsible Nutrition,
found that 85% of American adults are confident in
the safety, quality and effectiveness of dietary supple-

Who and what was studied?

The researchers looked at 10 years of data (2004-2013) to
estimate the adverse events associated with dietary supplements in the United States from 63 different hospitals.
The selection of these hospitals was meant to be nationally representative and included locations that had
24-hour emergency departments. Trained patient record
abstractors reviewed the reports from each hospital to
identify cases where supplements had been implicated
as the likely source of the adverse event. These abstractors have been trained to analyze and compile medical
information contained in patient records.
33

Cases were scanned for emergency room visits where

ing intentional self-harm, drug abuse, therapeutic

the treating clinician had explicitly ascribed dietary

failures, nonadherence, and withdrawal.

supplements as the root cause of the medical issue. This

included herbal or complementary nutritional products
such as botanicals, microbial additives, and amino acids,
in addition to micronutrients like vitamins and minerals.
Products that may typically be classified as food were
excluded, like energy drinks and herbal tea beverages.
Topical herbal items and homeopathic products were
included in the analysis even though they do not fall
under the regulatory definition of dietary supplements.
Adverse events were classified as anything causing
adverse or allergic reactions, excess doses, unsupervised ingestion by children, or other events like
choking. Due to the non-standard death registration
practices among different hospitals, cases involving a
mortality were not included, as were any cases involv-

Researchers examined patient records from 2004 to

2013 from 63 different hospitals. Cases where the
treating clinician had identified a supplement as
the cause of the medical emergency were extracted
from the dataset. However, deaths associated with or
caused by supplements were not included, as hospitals differ in their practice of registering mortalities.

What were the findings?

Some of the major findings are summarized in Figure
1. Over 3,600 cases were identified within the predetermined 10-year period. The researchers extrapolated
from these data that the U.S. experienced an average

Figure 1: Supplement safety by the numbers

34

of 23,000 supplement-related emergency department

of ER visits (65.9%) were due to herbal or complemen-

visits per year, with estimates ranging from 18,600 to

tary nutritional products. The top five products in this

27,400. Of these 23,000 emergency room visits, it was

category included the following: weight loss (25.5%),

calculated that about 2,150 (9.4%) of these result in

energy (10.0%), sexual enhancement (3.4%), cardiovas-

hospitalization. About 88% of these ER visits were

cular health (3.1%), and sleep, sedation, or anxiolysis

attributed to a single supplement, as opposed to inter-

(i.e. anti-anxiety) (2.9%). Multivitamins or unspecified

actions or mixtures of multiple supplements. The

vitamin products were the biggest contributors to ER

average age of patients treated for supplement-related

visits under the micronutrient product category.

adverse events was 32 years, and the majority of these

cases were female.

ER visits also varied according to gender and age.

Weight loss and micronutrient supplements dispro-

Figure 2 shows age and supplement category related

portionately landed females in the ER, while sexual

results. About a quarter of ER visits involved people

enhancement and bodybuilding products largely affect-

between the ages of 20 to 34, but people older than 65

ed males. Among patients younger than four years old

years old were more likely to have a visit that resulted

and adults over 65, micronutrients were the number

in hospitalization. Of patients above 65 admitted to the

one cause of emergency department visits. This is in

ER, 16% had to be hospitalized. Surprisingly, one-fifth

contrast to the other age groups, where herbal and

of supplement-related ER visits were due to accidental

complementary nutritional products were the biggest

ingestion by children. When the data covering unsuper-

contributor. In people ages five to 34, weight loss prod-

vised ingestion of dietary supplements by children was

ucts or energy products were implicated in more than

not included, the researchers found that the majority

50% of ER visits. Weight loss products mostly affected

Figure 2: Summary of which types of supplements lead to ER visits by age

Source: Geller AI et al. N Engl J Med. 2015 Oct.

35

patients from 20 to 34 years of age, while the micro-

ed dietary supplements as fundamentally healthy. That is,

nutrients iron, calcium, and potassium mostly affected

the general public overwhelmingly perceives these prod-

those older than 65.

ucts to be safe and effective, but the present data does not
support this notion (ERD readers excluded. We think

About 23,000 people go to the ER for supplement-related visits every year. The biggest contributors to
this are herbal or complementary nutritional products like weight loss and energy supplements, which
largely affect people between the ages of five to 34.
Females are more likely than males to end up in the
ER due to adverse supplement reactions. Those over
the age of 65 are most at risk for an ER visit due to
micronutrient supplements such as iron, calcium,
and potassium.

you are all ahead of the curve on this one).

However, it should also be noted that overall incidences
of supplement-related ER visits have remained constant over time. No significant changes were detected
between 2004 and 2013 when accounting for population increases. The only increase that occurred was
ER visits associated with micronutrient supplements,
which jumped 42.5%, from 3,212 to 4,578 cases in this
same time frame.
Unlike their highly regulated pharmaceutical coun-

What does the study really

tell us?
While 23,000 annual supplement-related emergency visits may sound high, this is less than 5% of pharmaceutical
product-related ER visits. However, these ER admittance
rates do not line up with the marketing that has promot-

terparts, there are no legal requirements for dietary

supplements to identify any potential adverse effects or
major drug interactions on their packaging. The lack of
adequate warning labels may be a contributing factor
to why histories of dietary supplement usage are rarely
obtained by clinicians. This can be due to a combination of clinicians not asking proper patient screening
questions and to a lack of disclosure by the patient.

Proprietary Blends
The FDA has established labeling standards dictating what must appear on a supplements
packaging. Manufacturers must list out each ingredient, and are required to display the amount
or percentage of daily value of those ingredients.
A proprietary blend falls under a slightly different set of regulations. Blends are a unique mixture of ingredients that are typically developed by the manufacturer. The FDA requires that all
ingredients of a proprietary blend be listed on the label in descending order according to predominance of weight. While the amount of the blend as a whole must be listed, the amount of
each ingredient included in the blend does not.
Blends are used to help prevent the competition from knowing what the specific formulation is.
But it can also hide the fact that very little of an active ingredient may be in the bottle. So while
a proven performance enhancing ingredient like creatine may be listed in a proprietary blend, it
could be well below what is considered to be an effective dose.
36

Given that there is a tendency to underreport sup-

In light of this lack of regulatory oversight, if you are

plement usage, the researchers have noted that their

currently taking or thinking about adding a supplement

calculations of emergency department visits attributed

to your diet, be sure to notify your doctor. Supplements

to supplement-related adverse events are probably an

can interact with prescription medication or could

underestimation. A further limitation was the relative-

exacerbate certain medical conditions. Warfarin

ly small sample of hospitals used. But this method of

(Coumadin) is a good example. It is a blood-thinning

data collection is likely to yield more accurate results

medication that can be prescribed to people at risk of

over voluntary reporting despite the fact that volun-

forming blood clots. To ensure that the medication

tary reporting would have likely allowed for a larger

works properly, these patients are usually placed on a

sample population.

low vitamin K diet, as vitamin K plays an essential role

in forming blood clots. If these patients do not disclose

While 23,000 annual supplement-related emergency visits may not be a large contributor to ER
visits in the larger scheme of things, it does provide
a counter-narrative to the marketing that often

that they are taking a multivitamin with vitamin K,

multivitamins being one of the most commonly used
supplements, they could be putting themselves at risk
for developing unwanted clots.

portrays supplements as always health promoting. Supplements are not required to come with

Currently, the supplement industry is partially

labels warning of adverse events or potential drug

policed by itself. Companies that market and sell

interactions, which can be a contributing factor to

supplement products do not have to show the FDA

supplement-related ER visits.

data of safety or efficacy in the same fashion that

pharmaceutical companies do. The FDA can step in

The big picture

The supplement industry is the wild west of nutrition.
By and large, DSHEA has hampered the ability of the
FDA to adequately regulate supplements. If you have
ever taken a supplement that makes a health claim,

when a supplement has been shown to cause harm

and pull it from the market. It is important to discuss all supplements you may be taking with your
doctor to avoid unpleasant or dangerous interactions.
Be sure to tell them even if they do not ask during
your screening.

you may have encountered this statement on the label:

These statements have not been evaluated by the Food
and Drug Administration. This product is not intended

Frequently asked questions

to diagnose, treat, cure, or prevent any disease. While

Is there any way to ensure that Im purchasing a quality

all ingredients must be declared on the label, there is lit-

supplement??

tle oversight to ensure that these ingredients are present

There are companies out there that do supply third-par-

in the supplement, at the doses that are advertised on

ty certifications to supplement manufacturers. These

the packaging. Under DSHEA, there is no requirement

companies will verify that the supplements listed on

for companies to provide any data to the FDA showing

the ingredient list are present in the concentrations

that their supplement is safe and effective, unless they

claimed. There are four major companies that provide

are introducing a new or novel ingredient. It falls on

these certifications, which are shown in Figure 3: NSF

the FDA to show that a supplement is unsafe before any

International, Informed Choice, Consumer Lab, and

action can be taken.

U.S. Pharmacopeia. With the exception of Consumer

37

Lab, all of these third-party certifiers print their seal on

related deaths were not included in the ER visit pro-

the products they have screened.

jection, which could lead to an underestimation, it is

also possible that emergency department physicians

The testing process often involves looking at the puri-

may have incorrectly ascribed certain signs and symp-

ty, strength, and bioavailability of the product. Good

toms to supplements, which could consequently lead to

manufacturing practices, which help to provide systems

overestimation. Essentially, the 23,000 annual ER visits

that track proper design, monitoring, and control of the

should be viewed as a very rough estimation.

manufacturing process and facilities, are also frequently

taken into account. Many employ continuous random

If you are currently taking or planning to introduce

testing in order for a given supplement to remain cer-

a supplement to your diet, be sure that you are con-

tified. It is very important to note that these companies

suming the recommended dose for that product and

do not test for efficacy. That is to say, these certifications

consult your doctor before hand. Supplements are not

do not ensure that any health claims made about the

automatically beneficial for health, no matter what the

supplement are truthful.

marketing says. Treat dietary supplements the way you

would treat medication, with caution and respect for

What should I know?

their ability to both help and harm your health.

While 23,000 dietary-supplement related ER visits may

not seem like a lot when compared to something like

An incredibly effective supplement may also be incred-

the 610,000 deaths caused by heart disease every year

ibly harmful given the right (well wrong) context.

in the U.S., it is something that can be easily prevented

Talk about the under-discussed issue of supplement

with education and awareness. Although supplement

safety at the ERD Facebook forum.

Figure 3: Third-party supplement certifications

38

In closing...
Thanks again for reading ERD. We enjoy helping people stay up to date on research,
whether youre dietitians, trainers, physicians, or simply people interested in improving your health.
Click here to learn more about how Examine.com evolved over the past five years.

ERD is about as thoroughly peerreviewed as you can get, outside of an

academic journal. And having peerreviewed for major journals, I can say with
certainty that we take more care in fact
checking and idea bouncing than many
of them. For this, each of the nineteen
regular ERD contributors are incredibly
proud, and we strive to improve on that
each and every new issue.
- Kamal Patel

Kamal Patel, Editor-in-Chief

39

Credits
Copy Editor: Dmitri Barvinok
Infographics: Antonius Khengdro, Hieu Nguyen, Jessie Alley & Calla Lee
iStock.com/ALEAIMAGE
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40

The espresso
effect: caffeine and
circadian rhythm

Effects of caffeine on the human

circadian clock in vivo and in vitro

41

Introduction
Caffeine has been a component of the human diet for

mal circadian rhythm of melatonin production, and

evaluated the cellular mediators of any potential effect.

centuries, primarily through the consumption of coffee

and tea. Culturally, coffee and tea are two of the main

Research has backed the commonly seen experience

beverages that tie together many human interactions.

of caffeine late in the day disrupting sleep and hence

Chemically, caffeine is well-known for being an ade-

circadian rhythms. The specific mechanisms for this,

nosine receptor antagonist that blocks the action of

as well as impact on the sleep hormone melatonin,

adenosine. Because adenosine mediates the perception

are not fully understood though.

of drowsiness, caffeine consumption results in alertness.

A circadian rhythm is essentially an organisms daily
internal clock. It is not restricted to humans and exists
in most living things, even bacteria. For us, the circadian clock follows a roughly 24-hour cycle, responding
primarily to light and darkness in our environment, and
acting to regulate countless bodily functions. One of the
most important hormones in this context is melatonin,
which is released by the pineal gland in the brain and is
involved with sleepiness and sleep regulation.
Melatonin is secreted in response to darkness to help
prepare the body for sleep. It is well-established that
exposure to bright light suppresses melatonin production. However, the ability of a stimulus to interfere with
normal circadian function depends on the time of day.
A pulse of blue light before bed will indeed suppress
melatonin production and delay sleep, but that same
pulse of light at noon will have no effect because melatonin production is low at noon anyway.
However, one recent population-based study of adults
estimated that 90% of individuals consume caffeine
in the afternoon (12:00-6:00 pm) and 68.5% of people consume caffeine in the evening (6:00 pm - 12:00
am). Similarly, 37% of high schoolers who consume
caffeine report not using it until after 5:00 pm. Doseresponse studies in humans and animals demonstrate
that increasing doses of caffeine administered at or near
bedtime are associated with significant sleep disturbance. Thus, the current study attempted to test whether
nighttime caffeine consumption would delay the nor-

Who and what was studied?

Five healthy young participants (three female and
two male) who maintained regular eight-hour sleep
schedules were recruited to undergo four experimental
sessions to test how caffeine, bright light, or both affected their circadian rhythms. After seven days of being
monitored in their daily lives to make sure they maintained a regular sleep schedule, the participants moved
into a research environment that was free of external
time cues for five days.
On day three of the laboratory stay, the participants
consumed a pill containing rice flour (placebo) or caffeine (2.9 mg/kg, approximately equal to the caffeine
found in a double espresso) in a double-blind manner about three hours before their habitual bedtime.
Starting at bedtime, the participants then sat under
ceiling-mounted fluorescent lamps providing either
broad-spectrum white-light similar to modest sunlight
(about 3000 lux) or dim-light similar to moonlight
(about 1.9 lux). All procedures were repeated four
times over roughly 49 days so that each participant
experienced four experimental sessions: placebo +
dim-light; placebo + bright-light; caffeine + dim-light;
and caffeine + bright-light. Saliva, collected for melatonin assessment, was taken every 30-60 minutes to
determine how each of these conditions affected the
circadian rhythms of the participants.
The participants abstained from over-the-counter med-

42

ications, supplements, and caffeine for two weeks, naps

for one week, exercise for three days, and alcohol for

There were only five study participants, all of whom

two days before and throughout the experiment.

were healthy. They spent a month and a half under

controlled sleep routines: taking a caffeine pill three

To more specifically understand the effects of caffeine at

hours before bedtime, then being exposed to either

the cellular level, the circadian transcriptional rhythms

bright or dim light. Melatonin secretion was estimat-

were studied in vitro in a well-understood human cell

ed, and caffeines cellular effects were assessed in a

line. This cell line expressed all of the proteins needed

separate in vitro experiment.

to assess caffeines effects on circadian rhythms, especially adenosine receptors, phosphodiesterases, and
ryanodine receptors.

How exactly might caffeine screw up my circadian rhythm?

The circadian clock in humans is controlled primarily by the suprachiasmatic nucleus (SCN) within the
brain. The SCN is a group of cells that sit by the
optic nerve and respond to light and other stimuli
that come in from the eye. Hypothetically, caffeine
could affect the SCN (and other tissues throughout
the body) in three ways, depicted in Figure 1.

reasons. One reason is that caffeine blocks adenosine receptors that normally reduce cAMP
levels. Secondly, caffeine binds phosphodiesterase
enzymes that act to degrade cAMP, and when these
enzymes are blocked, cAMP levels are raised. Its
also been shown that circadian rhythms within the
SCN are regulated in part by the release of calcium
ions during the stimulation of ryanodine receptors,
The first is through increasing a cells levels of which caffeine also binds. Thus, caffeine has three
cyclic AMP (cAMP), which is often created inside primary ways in which it can interact with the SCN
a cell in response to a signal. This occurs for two to influence circadian rhythms.

Figure 1: Possible ways caffeine could affect circadian rhythm

43

What were the findings?

The study results are summarized in Figure 2. Bright
light, caffeine, and their combination significantly
delayed the onset of melatonin production compared
to the dim light plus placebo condition. Specifically,
dim light plus caffeine delayed production by about 40
minutes, bright light and placebo by 85 minutes, and
bright light exposure plus caffeine by 105 minutes. The
difference between bright light plus caffeine and dim
light plus caffeine was also statistically significant, and
there was a trend for bright light in combination with

placebo to be greater than dim light combined with

caffeine. Collectively, these results indicate that bright
light and caffeine both independently interfere with the
normal onset of melatonin secretion, with bright light
having a stronger effect that is not significantly modified by caffeine.
There were marked inter-individual differences in the
response to caffeine ingestion. When caffeine was
ingested under dim light conditions, two of the five
participants experienced no delay of melatonin secretion, two were delayed by 30-60 minutes, and one was
delayed by nearly two hours. This variability was also

Figure 2: Results from this study

observed under the bright light caffeine condition, with

phase delays ranging from 45-180 minutes. In contrast, the effect of bright light only was more clustered
around phase delays of about 60-100 minutes. This
variability could be the result of individual differences
in caffeine metabolism, but this study didnt evaluate
such differences.
There was also a significant positive linear association
between the phase shifts induced by dim light with
caffeine and bright light with caffeine as well as dim
light placebo and bright light placebo. This suggests
that the gradual phase shift that occurs naturally, due
to the fact that many peoples circadian rhythm is longer than 24 hours, correlates to the strength of the light
that affected their sleep cycles. In other words, the more
a persons sleep cycle drifts naturally, the more light
affected it.
Finally, experiments in human cells showed that caffeine delays the circadian rhythm by acting on the
A1-type adenosine receptor to increase cellular levels
of cAMP rather than affecting phosphodiesterases and
ryanodine receptors. Therefore, this may be the mechanism responsible for the phase delay observed in the
study participants.

44

depends on the time of day. The caffeine in this study

Consuming caffeine three hours before bedtime

was consumed three hours before habitual bedtimes in

delays the circadian clock by blocking adenosine

an environment that was free of light cues. It stands to

receptors. The magnitude of the effect is about half

reason that caffeine may not be as detrimental when

that observed with bright light alone, and combin-

consumed later in the day during the summer season,

ing caffeine with bright light showed a larger delay

compared to the winter season, as there is more sun-

than either alone, although the difference wasnt

light at later times of day. Still, even during winter, the

statistically significant. There were marked individ-

advent of modern technology and blue light may very

ual differences in the response to caffeine ingestion,

well trick our bodies into believing it is sunny out. How

while the effect of bright light exposure was similar

caffeine interacts with environmental cues remains to

in all participants.

be elucidated.

What does the study really

tell us?
This study had three main takeaways: caffeine delays
melatonin secretion when consumed three hours before
sleep, there are marked individual differences in the
magnitude of this effect, and caffeine delays the cellular
clock through the blocking of adenosine receptors that
subsequently increased cAMP in the cell.
The dose and timing of caffeine in the study are two
limitations, especially in light of the individual differences in response to caffeine. The amount of caffeine
used in this study was 2.9 milligrams per kilogram of
bodyweight, which equates to roughly 240 milligrams
for a 180 pound person. Black and green teas contain
14-70 milligrams of caffeine per eight fluid ounces (one
cup) and brewed coffee contains 95-200 milligrams.
Thus, unless someone is used to downing energy drinks
or having a double espresso for dessert, the dose of
caffeine used in this study was relatively high compared
to real life scenarios. Future research could test smaller
doses to determine if a threshold level for intake exists.
Even so, two of the five participants showed no phase
delay with the current dose, suggesting that the effects
of caffeine may ultimately depend on the individual.
As mentioned in the introduction, the ability of a
stimulus to interfere with normal circadian function

In a study using a more real-life research setting,

healthy day workers popped a 400 milligram caffeine
pill zero, three, or six hours before their typical bedtime.
This study showed that timing does not differentially
impact overall sleep disruption. Caffeine is metabolized
mostly by the P450 enzyme system within the body
(specifically the CYP1A enzymes). This is the bottleneck step in clearing caffeine from the blood and it is
subject to genetic variance, which could explain the
variability observed in the current study and possibly
the results of this aforementioned study as well. People
who are fast caffeine metabolizers may be able to
consume caffeine closer to bedtime without influencing
their circadian rhythms, compared to slow metabolizers. If youre curious, data from commercially available
genetic tests can tell you what kind of caffeine metabolizer you are.
The finding that caffeine acts on a cellular level via the
adenosine receptor to activate cAMP lends support
to the hypothesis that dynamic changes in cAMP signaling regulate circadian rhythms. The current study
therefore suggests that caffeine and other compounds
that bind and inhibit adenosine A1 receptors would
have similar effects. Other mechanisms for caffeines
effect cannot be ruled out, however.
The human portion of this experiment used a very
tightly controlled design that may have substantially
45

limited the number of participants, which came in at a

shift work that involves circadian disruption has been

meager five people. The laborious nature of sleep stud-

labeled as a probable carcinogen by the International

ies makes this type of research difficult. Nonetheless,

Agency for Research on Cancer.

future trials with larger sample sizes will be needed to

confirm these findings, as well as extend the data from

Very rarely, such as in the laboratory, would we be

just melatonin to actual sleep onset and wake times.

deprived of environmental time cues that entrain

our circadian rhythm. However, the modern era has

The variance in caffeine metabolism shown in previous research, specifically due to the CYP1A enzymes,
may explain why individual results varied so much
in this experiment. This study also suggests that out
of the ways in which caffeine may impact circadian
rhythms on a cellular level, acting on the adenosine
receptor may be the most significant.

brought with it wildly altered zeitgebers (German for

time giver; the term used to denote circadian rhythm
cues) that may interrupt the natural light/dark cycle,
the most notorious being devices that emit blue light.
This includes smartphones, laptops, televisions, and
most modern light bulbs. In ERD Issue #4 (Feb, 2015),
The iPad Hangover discussed research that showed that
people using iPads before bed took about ten minutes longer to fall asleep, experienced less REM sleep,

The big picture

Sleep is a fundamental biological process for most
organisms. Humans spend, on average, about onethird to one-fourth of their lives asleep. There are many
hypotheses for the purpose of sleep, but it is clear that
sleep deprivation and a dysregulation of normal circadian rhythms are linked to disease: cancer, metabolic
syndrome, type-2 diabetes, and even death. In fact,

and found it much harder to feel fully awake in the

morning than people that read print books before bed.
Importantly, it appears that bright light exposure duration is a more influential variable on the human clock
than bright light intensity, at least at bright light levels
replicating various daytime conditions.
The current study covers another zeitgeber caffeine,
and shows that it can influence the circadian clocks of

[...] people using iPads before bed

took about ten minutes longer to fall
asleep, experienced less REM sleep,
and found it much harder to feel fully
awake in the morning than people
that read print books before bed.
46

individuals when consumed three hours before bed.

Moreover, the study shows that these effects occur on
a cellular level but are subject to individual differences
in caffeine metabolism. Other research has supported these findings by showing that caffeinated tea and
coffee beverages consumed throughout the day had a
dose-dependent negative effect on the time it takes to
fall asleep, the time spent sleeping, and sleep quality.
Caffeine and bright light exposure are not the only
lifestyle and environmental variables to affect the
human clock. It is not uncommon for many people to
exercise after work and several hours before going to
bed. Yet, one small study in young, fit males showed
that exercise delays the onset of melatonin production
just like caffeine and bright light do, and the delay is
more pronounced the closer to bedtime the exercise is
performed. This may be due to the stress response of
exercise and rise in cortisol levels that appear to precede the suppression in melatonin. On the other hand,
exercising in the morning with sun exposure has been
shown to have positive effects on sleep-related hormonal responses, sleep habits, and quality of sleep.
Finally, some research suggests that drinking alcohol at
night can suppress melatonin production with doses as
little as 0.5 grams per kilogram of bodyweight, or about
2.5 cans of beer for a 160 pound person. Smoking may
also reduce melatonin levels, albeit only in response to
melatonin supplementation (which is not uncommon)
rather than natural production.

Poor sleep is linked to most of the major causes of

death, so normalizing ones circadian rhythm may
be one of the highest-return health habits possible.
While this study focused on two specific zeitgebers,
light and caffeine, others such as exercise (specifically late in the day), alcohol, and smoking can also
impact circadian rhythms.

Caffeine and
bright light
exposure are
not the only
lifestyle and
environmental
variables
to affect
the human
clock. It is not
uncommon for
many people
to exercise
after work and
several hours
before going to
bed.
47

Frequently asked questions

How can I maximize my sleep quality?

What should I know?

Caffeine isnt just used a morning pick-me-up. Many

As we just learned, avoid blue light and caffeine close to

people drink caffeine late into the afternoon and into

bedtime to maximize sleep quality. This sounds simpler

the evening, which this trial shows is a recipe for dis-

than it is in reality, as few people turn off all comput-

rupted melatonin synthesis. The study also showed

ers, tablets, and light bulbs at night (the light output of

some additive effect of consuming caffeine late in the

some of these sources is shown in Figure 3). If you have

day while being exposed to bright light at night, the

a dimmer for a light switch, that can come in handy. If

latter of which is quite common in modern society.

you really, really want to reduce or eliminate blue light

exposure and improve circadian rhythms, blue light

Luckily, the takeaway isnt complex: dont expose your-

blocking glasses are an option with research backing.

self to bright light or consume caffeine close to bedtime.

Though specific responses to caffeine can be highly

A quiet environment also helps, as loud noises can

variable, due to individual genetic differences in its

disrupt sleep even if the sleeper doesnt wake up.

metabolism, sleep disruption has been strongly linked

Establishing a habitual bedtime can reduce the time

with so many diseases that when in doubt, go decaf.

it takes to fall asleep. As a last resort, there are also

supplemental options. Melatonin is probably the most
popular option and is safe for daily use. Lemon balm is
another option.

If youre reading this late at night, shame on you. But

knowing is half the battle, so get some sleep before
heading to the ERD Facebook forum to discuss the
world of zeitgebers and circadian rhythms.

Figure 3: Some rough estimates of lux values and caffeine amounts

48