REVIEW ARTICLE

Journal of

Basic Models Modeling

Resistance Training: An Update
for Basic Scientists Interested
in Study Skeletal Muscle
Hypertrophy

Cellular
Physiology

2
~
JASON CHOLEWA,1 LUCAS GUIMARAES-FERREIRA,
TAMIRIS DA SILVA TEIXEIRA,3
 3 ALICE LODETTI,3
MARSHALL ALAN NAIMO,4 XIA ZHI,5,6 RAFAELE BIS DAL PONTE DE SA,
MAYARA QUADROS CARDOZO,3 AND NELO EIDY ZANCHI3*
1

Department of Kinesiology Recreation and Sport Studies, Coastal Carolina University, Conway, South Carolina

Laboratory of Experimental Physiology and Biochemistry, Center of Physical Education and Sports,
Federal University of Espirito Santo, Vitoria, Brazil

Postgraduate Program in Health Sciences, Health Sciences Unit, Universidade do Extremo Sul Catarinense, Criciuma, Brazil

Division of Exercise Physiology, West Virginia University School of Medicine, Morgantown, West Virginia

Exercise Physiology and Biochemistry Laboratory, College of Physical Education, Jinggangshan University, Jinggangshan, PR, China

Exercise Physiology Laboratory, Department of Exercise Physiology, Beijing Sport University, Beijing, PR, China

Human muscle hypertrophy brought about by voluntary exercise in laboratorial conditions is the most common way to study resistance
exercise training, especially because of its reliability, stimulus control and easy application to resistance training exercise sessions at fitness
centers. However, because of the complexity of blood factors and organs involved, invasive data is difficult to obtain in human exercise
training studies due to the integration of several organs, including adipose tissue, liver, brain and skeletal muscle. In contrast, studying
skeletal muscle remodeling in animal models are easier to perform as the organs can be easily obtained after euthanasia; however, not all
models of resistance training in animals displays a robust capacity to hypertrophy the desired muscle. Moreover, some models of resistance
training rely on voluntary effort, which complicates the results observed when animal models are employed since voluntary capacity is
something theoretically impossible to measure in rodents. With this information in mind, we will review the modalities used to simulate
resistance training in animals in order to present to investigators the benefits and risks of different animal models capable to provoke
skeletal muscle hypertrophy. Our second objective is to help investigators analyze and select the experimental resistance training model
that best promotes the research question and desired endpoints.
J. Cell. Physiol. 229: 11481156, 2014. 2013 Wiley Periodicals, Inc.

In humans early training gains in muscle strength have been

regarded as the result of both neural and musculature
adaptations. Over the last half-decade several animal training
models have been developed as a way to increase both force
output and mass (hypertrophy) in the exercised muscle.
Contrary to the increases in maximal oxygen consumption
observed in animals with aerobic training using a treadmill,
measurements of maximal and submaximal force capacity in
vivo are complicated by several factors, including voluntary
capacity to perform resistance training, non-voluntary
electrical-based training under anesthesia, surgical
manipulation of muscles involved in the hypertrophic response,
and the utilization of positive or negative reward to stimulate
the animals to perform the exercise. Thus, the greatest
motivation for an animal to produce maximal capacity
voluntary muscular force in classic operant models is via direct
electrical stimulation to the brain, which is virtually impossible
to perform in subsequent experiments with the same animal
(Olds and Milner, 1954).
Pain avoidance has been demonstrated to be a greater
stimulus than food or water reward (Miller, 1951). According
to Timson (1990), the animal will perform a task only until the
effort involved in the task performance exceeds its desire for
2 0 1 3 W I L E Y P E R I O D I C A L S, I N C.

the stimulus. Thus, a model employing starvation as the main

stimulus will motivate the animal to exert only 5060% of its
maximal voluntary capacity, which will then negatively affect
muscular hypertrophy capacity either due to lack of overload
or nutrition. Therefore, we will first review animal models
employing non-voluntary maximal capacity force production as
a way to induce hypertrophy, and then discuss new methods
involving voluntary models. A summary of results of the models
reviewed is available in Tables I and II.

Dr. Jason Cholewa and Dr. Lucas Guimar~aes-Ferreira contributed

equally to this work.
*Correspondence to: Nelo Eidy Zanchi, Av. Universitaria, 1105
Bairro Universitario, C.P. 3167 | CEP 88806-000, Criciuma/Santa
Catarina, Brazil. E-mail: neloz@ig.com.br
Manuscript Received: 12 December 2013
Manuscript Accepted: 16 December 2013
Accepted manuscript online in Wiley Online Library
(wileyonlinelibrary.com): 25 December 2013.
DOI: 10.1002/jcp.24542

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25
RF, rectus femoris; EDL, extensor digitorum longus; SOL, soleus; FHL, flexor hallucis longus; PLA, plantaris; GAS, gastrocnemius; CSA, cross-sectional area.

2012
2002
Scheffer et al.
Fluckey et al.

JOURNAL OF CELLULAR PHYSIOLOGY

Non-Voluntary Non-Electric Exercise-Induced

Enlargement in Animal Models

12 weeks
4 weeks
Alternate days

43 steps

5 weeks
8 weeks
8 weeks
Every 2 days

80

1.1 m
2012
2003
2004
Dela Cruz et al.
Wirth et al.
Hornberger

Jump in a PVC cylinder containing water

Operant conditioning (progressive lift)
Ladder climbing (with progressive
load attached to the tail)
Ladder climbing
Flywheel resistance training

2009
1999
Zanchi et al.
Tamaki et al.

Plantar flexion of ankle joint

Squat Like Exercise

15 jump sessions
1215
1

Increase in PLA weight

Hypertrophy of GAS and PLA and increase
in the number of muscle fibers
Increase in EDL and SOL CSA
Increased performance
Increase in FHL weigth and total and
myofibrillar protein content

Attenuation of hindlimb suspension-induced

muscle atrophy in SOL
16
6575% 1 RM
12 weeks
12 weeks

8 weeks
36 weeks

Every third day

In the morning, at noon,
and in the evening
(Monday and Tuesday,
Thursday and Friday)
Three times/week
45 days/week
1m

2004
1988
Lee et al.
Klitgaard et al.

Ladder climbing IGF-I adenovirus

Plantar flexion of ankle joint

85

1215
26 weeks
4 days/week
1998
Duncan et al.

40 cm

Vertical

8 climbs or until failure

30 min of training
three times per day

Increase in EDL and SOL weights relative

to body mass and fibre hypertrophy
Increase in FHL weight
Increase in SOL and PLA weights

Increase in RF weight
8 weeks
5 days/week
90
40 cm

Progressive lift with loads attached

to the tail using a mesh
Ladder climbing
1990
Yarasheski et al.

Technical
Year
Author

TABLE I. Voluntary muscle hypertrophy models

Size/height

Angulation

Sessions/weeks

Training
period

20

Series/day

Muscle hypertrophy (%)

BASIC MODELS MODELING RESISTANCE TRAINING

One of the first methods to induce skeletal muscle

hypertrophy was developed by Thomsen and Luco (1944)
whereby a passive stretch applied to immobilized joints places
longitudinal tension upon the muscle (Alway et al., 1989;
Fig. 1F). Utilizing this model of overload Aoki et al. (2006)
reported an increase in sarcomeres in series leading to an
elongation of the target muscle. The application of rapamycin
was demonstrated to robustly suppress this response,
suggesting the mammalian target of rapamycin (mTOR)
pathway is involved in the longitudinal hypertrophy induced by
joint immobilization. This model of overload may be
appropriate to study skeletal muscle remodeling as a result of
stretch overload or joint immobilization; however, resistance
training in humans requires dynamic tension generation,
resulting in a force overload, and leading to the synthesis of
additional sarcomeres in series. Therefore, future investigators
sought to develop methods that more closely modeled
resistance training.
Goldberg (1968) developed an effective non-voluntary nonelectrically stimulated model (Fig. 1A) to induce skeletal muscle
hypertrophy through synergistic ablation (surgical removal of a
synergistic muscle, most often the gastrocnemius calcaneus
portion, generating overload and muscle hypertrophy of the
soleus and plantaris muscle). Although the use of this model to
mimic the effects of human strength training has been highly
criticized due to the surgical procedures (Taylor and
Wilkinson, 1986), McCarthy et al. (2011) demonstrated no
differences in muscle hypertrophy between mice with genetic
satellite cell depletion and non-depleted controls with 2 weeks
of synergistic ablation overload. Given the similar significant
improvements in muscle hypertrophy in both groups,
synergistic ablation remains an effective method to study
cellular signaling pathways leading to acute skeletal muscle
hypertrophy (Miyazaki and Esser, 2009).
On the other hand, because the targeted muscle is exposed
to a static stimulus (the animals bodyweight) the increase in
muscle mass occurs most rapidly during the first week of the
protocol and appears to reach a plateau 2 weeks following
surgery. Additionally, the animal is under constant overload
every time it moves, compared to separate training sessions
used in human resistance training or other animal models.
Thus, synergistic ablation cannot be used in long term studies
nor does it appear compatible with modeling the progressive
overload or periodization phases and nutrition schedules
required in human resistance training to induce maximal
changes in hypertrophy and strength.
Tenotomy is a technique where the gastrocnemius tendon is
detached and the synergistic muscle is placed under increased
muscle tension (Fig. 1A). Tenotomy appears less effective at
inducing overload and the resultant musculature hypertrophy
of the synergist (e.g., plantaris) when compared with surgical
ablation (Timson, 1990). Although the reason for the
difference is not clear, it appears that the cut tendon is able to
reattach when left intact within the muscle fascia. The critiques
of tenotomy are the same as those related to synergistic
ablation methods; however the magnitude of hypertrophy is
less and the possibility of the gastrocnemius tendon reattaching
the calcaneus tendon.
The use of chronically restricted venous blood flow was first
reported by Kawada and Ishii (2005) to induce skeletal muscle
hypertrophy in rats. This model does not involve exercise;
rather, blood flow to the hind limbs is diminished via a surgical
intervention. Fourteen days following the operation the
plantaris muscle increased in dry weight by 10% and the
concentration of myofibrillar protein increased by 23%.
Additionally, levels of nitric oxide synthase and the muscle

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TABLE II. Involuntary muscle hypertrophy models

Author

Year

Goldberg et al.
Goldberg et al.
Wong and Booth

1968
1975
1988

Baar and Esser

1999

Goldspink

1999

Kawada and Ishii

Haddad et al.

Technical

Period

Estimulus

Surgical ablation of GAS

Tenotomy of GAS
Weight-lifting exercise

6 days
14 days
16 weeks

Walk
Walk
Electric external load

6 weeks

Electric

4 days

Electric

Increase in TA wet weight

2005

Surgical implantation of electrodes

and electrical stimulation
Stretch combined with eletrical stimulation
of anterior tibialis muscle of adult rabbit
Chronic restriction of blood flow to muscle

Increase in SOL and PLA weights

Increase in SOL weight
Increase in GAS we weight and
protein content
Increase in EDL and TA wet weights

Venous occlusion

2006

Electrical stimulation

Increases in PLA dry weight body weight

and myofibrillar protein content
Attenuation of hindlimb suspension-induced
muscle atrophy in GAS (muscle weight)

2 weeks
6 days

Isometric contractions

Muscle hypertrophy (%)

EDL, extensor digitorum longus; SOL, soleus; TA, tibialis anterior; flexor hallucis longus; PLA, plantaris; GAS, gastrocnemius.

insulin like growth factor-1 (IGF-1) also increased. It is difficult

to speculate on the level of difficulty or safety of this model as a
detailed description of the surgery is not completely available in
the literature; however, this model appears to be consistent
since Kawada and Ishii (2008) reproduced the results of the
first study and also reported decrements in type I muscle fibers.
Although plantaris hypertrophy was modest compared to
synergist ablation, chronic blood flow restriction may be a
novel model to study hypertrophy in animals. When translating
the results to human training two questions arise: (1) What are
the effects of chronic blood flow restriction combined with
muscular tension? (2) Does blood flow restriction occurring
for longer than 2 weeks compromise the health of the animal or
result in a plateau in muscle hypertrophy? Given that
intermittent blood flow restriction under low tension
phosphorylates P70S6K and muscular hypertrophy in humans
(Fujita et al., 2007), answering these questions are essential to
evaluating the ability to translate this model to human
resistance training.
Non-Voluntary, Electric Exercise-Induced Enlargement
in Animal Models

Wong and Booth (1988) developed a novel non-voluntary

model to load the hind limb and induce muscle hypertrophy. In
this model the animal is anesthetized, the foot is attached to an
immovable metal plate with adhesive tape, and muscular
contraction is stimulated electrically with joint of the animal
starting in a neutral position (Fig. 1E). The ability of this model
to induce hypertrophy and increased muscle fiber cross
sectional area is inconsistent and produces only modest
results; however, using a modified model, Baar and Esser
(1999) demonstrated P70S6K phosphorylation and
polyribosome formation, which indicates that the Wong and
Booth model is capable of increasing protein synthesis.
Goldspink (1999) modified the protocol proposed by Wong
and Booth (1988) by loading the limb in a stretched position
(elongation) and allowing for the electrical stimulus to induce a
dynamic contraction (Fig. 1I). This combined model resulted in
a greater increase in protein synthesis compared to the
elongation model or isometrically loaded models alone.
Moreover, using the combination of elongation and dynamic
overload Goldspink demonstrated the activation of a transcript
derived from the IGF-1 local to skeletal muscle, which has been
labeled mechano growth factor (MGF). MGF presents an insert
with 52 base pairs in the E domain of the gene, which alters the
reading frame of the 30 end, resulting in satellite cell
proliferation/activation following muscle damage, ultimately
leading to muscular repair and hypertrophy (Hill and
Goldspink, 2003). This model allows the researcher to apply an
identical maximal pulse to generate maximal tetanic force, and
thus eliminates the need to readjust the electrical stimuli.
Although the combination of muscular elongation and nonJOURNAL OF CELLULAR PHYSIOLOGY

voluntary contraction may be viable in studying acute increases

in protein synthesis, electrical pulses under anesthesia are
difficult to perform, as is the ability to apply a consistent,
progressive increase in electrical stimulation to match an
increased load required to induce hypertrophy.
Resistance Training (RT) Exercise Under Unloading
Conditions

Another interesting resistance training model was presented

by Haddad et al. (2006) whereby rats were unloaded via hind
limb suspension (HS) to induce muscular atrophy for 6 days.
Animals in the resistance training group (HST) were trained
every other day. Briefly, animals were anesthetized and
stimulation electrodes consisting of Teflon-coated stainless
steel wire were introduced into the subcutaneous region
adjacent to the popliteal fossa via 22-gauge hypodermic
needles. Wire placement was lateral and medial of the location
of the sciatic nerve allowing for field stimulation of the nerve.
The stimulation wires were then attached to the output poles
of a Grass stimulus isolation unit interfaced with a Grass S8
stimulator. This allowed for the delivery of current to the
sciatic nerve resulting in muscle contraction. The right leg was
positioned in a footplate attached to the shaft of a Cambridge
model Hergometer, adjusted to produce maximal isometric
tension. Each training bout consisted of a series of four sets of
contractions with 5 min of recovery between sets. Each set
consisted of a series of 10 maximal isometric contractions
lasting 2 sec each with 20 sec of rest in between contractions.
Thus each training session lasted for 27 min, during which the
muscle was activated for a cumulative time of 80 sec.
Compared with normal controls Haddad et al. (2006)
reported the gastrocnemius of the HS animals decreased 20%.
Although the RT program had a positive effect on maintaining
relative muscle weight at a higher level compared with the HS
group (8%), this response may in part have been due to edema,
as total protein concentration was slightly lower (7%) in the
HST compared with the HS group. This response
demonstrates the negative impact of unloading on the hind limb
musculature by illustrating that the myofibril pool was indeed a
primary target of the atrophy response. The results of this
study suggest that the process of muscle atrophy is not
opposite of muscle hypertrophy, and demonstrate the inability
of isometric based RT to spare muscle protein during
unloading. Therefore, although an isometric model of RT may
be appropriate to induce hypertrophy, researchers using
resistance training in animal models of diseases (i.e.,
dexamethasone-induced diabetes; Nicastro et al., 2012a)
should consider performing experimental pilot studies with
dynamic based contractions prior to data collection.
On the other hand, Fluckey et al. (2002) demonstrated that
dynamic resistance training is capable of preventing muscle
wasting during unloading. In this model, Fluckey et al.

BASIC MODELS MODELING RESISTANCE TRAINING

Fig. 1. Progression of the development of resistance training models in Rodents (19682012). (A) Surgical ablation; (B) Tenotomy; (C)
voluntary plantar extension; (D) 85 weighted ladder climb; (E) non-voluntary hind-limb extension; (F) passive stretch; (G) 90 weighted ladder
climb; (H) electric stimulated squat; (I) modified non-voluntary hind-limb extension; (J) modified flywheel with hind-limb suspension; (K)
operantly conditioned squat; (L) modified operantly conditioned squat. (M) jumping submersed in water with overload. Adapted by the
authors.

JOURNAL OF CELLULAR PHYSIOLOGY

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developed a modified version of the human flywheel resistance

exercise apparatus so rats could be trained while in hind-limb
suspension. This poses a major advantage over the model used
in Haddad et al. as the animals can be trained with dynamic
resistance exercise independent of gravity and without being
removed from the cage. Briefly, a rat is tethered via a leather
and velcro vest attached to a nylon cord and spooled around an
inertia wheel located on the outside of the resistance exercise
apparatus. The rat is allowed to place its feet on a shock grid
suspended at the top of the apparatus (to accommodate the HS
state) and an illumination bar capable is located in the apparatus
opposite to the shock grid. The bar is then illuminated which
results in a repetition by the animal. The movement is similar to
squats as performed by humans, as extension occurs at the hip,
knee and ankle joints. When required a shock is applied briefly
(<1 sec) to stimulate the movement of the animal. In this model
the resistance training protocol consists of 11 exercise
sessions over a 4-week period, with two sets of a maximum of
25 repetitions (or point of failure; that is, the animal would not
respond to the illuminated bar even with a brief foot shock) for
each session.
In a recent study Fluckey et al. (2002) employed a 4-week
period of HS with three training sessions per week, each
session lasting approximately 15 min (Fig. 1J). In contrast to
Haddad et al. (2006) the flywheel resistance protocol resulted
in an increase in soleus mass and body mass compared to HS
alone. Moreover, an increase in MPS and a muscle protein
sparing effect of 50% was observed compared to HS alone,
demonstrating the effectiveness of dynamic based resistance
training over isometric based resistance training in a state of
unloading. Interestingly, the ELD muscle was not affected by
flywheel resistance training, which arouses muscle recruitment
questions utilizing this resistance training model. It should be
noted that the 25 maximal repetitions used in this study did not
result in dramatic muscle hypertrophy, and thus researchers
using this protocol to investigate hypertrophy based research
questions should consider increasing the volume to provide
adequate overload in order to stimulate muscle hypertrophy
during periods of unloading.
Voluntary Exercise-Induced Enlargement in Animal
Models

Yarasheski et al. (1990) investigated whether heavy-resistance

exercise training alters the skeletal muscle fiber composition
of young rats. To model resistance exercise the rats where
trained to climb with weights attached to the rats tail a 40-cm
90 mesh incline 20 times/day 5 days/week for a food reward.
The load was progressively increased from body weight the
first 5 days, to 60 g on day 6, and then 30 g was added every
3 days thereafter (Fig. 1G). The researchers observed a
pronounced change in the muscle phenotype and a change in
the muscle mass of trained animals, but only in the superficial
region of the rectus femoris, which contained a greater
proportion of type, IIb fibers compared with the deep region. In
contrast, Duncan et al. (1998) trained male Wistar rats (3
weeks old) to climb a 40-cm vertical ladder (4 days/week) while
carrying progressively heavier loads secured to their tails. After
26 weeks of training the rats were capable of lifting up to 800 g
or 140% of their individual body mass for four sets of 1215
repetitions per session; however, no difference in body mass or
absolute extensor digitorum longus (EDL) and soleus mass was
observed between the trained rats and age-matched sedentary
control rats. Absolute and relative heart mass was greater in
trained rats than control rats, and when expressed relative to
body mass, the mass of the extensor EDL and soleus muscles
were greater in trained rats than control rats. Despite an
increased ability of the rats to lift progressively heavier loads,
this heavy resistance training model did not induce gross
JOURNAL OF CELLULAR PHYSIOLOGY

muscle hypertrophy nor did it increased the force-producing

capacity of the EDL or soleus muscles. The discrepancy in
results between Duncan et al. (1998) and Yarasheski et al.
(1990) was likely due to the muscles sampled and measured.
The soleus is predominantly type I muscle fiber and likely did
not suffer enough overload to induce hypertrophy. We suggest
researchers using the ladder climb model to study hypertrophy
or molecular signaling in protein synthesis evaluate samples
from muscles with a higher proportion of type II fibers, such as
the rectus femoris or gastrocnemius.
This model of mesh scale was then modified into a second
one where SpragueDawley rats were trained to climb a 1.1-m
vertical (80 incline) ladder with weights secured to their tail
(Hornberger and Farrar, 2004). The rats were trained once
every 3 days for 8 weeks. Each training session consisted of 49
(6.02  0.23) climbs requiring 812 dynamic movements per
climb. Based on performance, the weight carried during each
session was progressively increased. Over the course of
8 weeks, the maximal amount of weight the rats could carry
increased 287% and the improved training performance was
associated with a 23% absolute increase in the weight of the
flexor hallucis longus (FHL), with a concomitant 24% increase in
both total and myofibrillar protein. On the other hand, Scheffer
et al. (2012) analyzed oxidative stress in skeletal muscles using a
similar model of climb ladder (43 steps) in four different
resistance training protocols: Muscular resistance training: RT
consisted of climbing the ladder carrying a load of 10% of body
weight, which was progressively increased to 20%, 30%, 40%,
and 50%, three to six sets with 2-min breaks, and 1215
repetitions. Hypertrophy training: HT consisted of climbing the
ladder carrying a load of 25% of body weight, which was
progressively increased to 50%, 75%, and 100%, three to six
sets with a 2-min break and 810 repetitions. Strength training:
ST consisted of climbing the ladder carrying a load of 25% of
body weight, which was progressively increased to 50%, 100%,
125%, 150%, 175%, and 200%, three to six sets with a 2-min
break, and three to five repetitions (Fig. 3). After 12 weeks
of training on alternate days, body weight was not different
amongst groups and the red portion of the brachioradialis was
removed and oxidative parameters were assessed. Although
muscle hypertrophy was not measured, HT caused an
imbalance in oxidative parameters in favor of pro-oxidants,
leading to oxidative stress in skeletal muscle.
In a related study Lee et al. (2004) tested whether adenoviral
administration of IGF-I (rats were injected with recombinant
AAV harboring rat IGF-I cDNA (rAAVIGF-I) was capable to
increase FHL muscle mass. Using the ladder climb model
(1-m ladder with 2-cm grid steps and inclined at 85), 8 weeks
of resistance training, a 23.3% increase in muscle mass was
observed in the FHL (Fig. 1D). Viral expression of IGF-I without
resistance training produced a 14.8% increase in mass and the
combined interventions produced a 31.8% increase in muscle
mass. Therefore, the combination of resistance training and
overexpression of IGF-I induced greater hypertrophy than
either treatment alone. These results suggest that a
combination of resistance training and overexpression of IGF-I
could be synergistic and can improve muscle hypertrophy
through adenoviral transfections. It must be remembered
that this original finding was revolutionary at that time and
generated a lot of knowledge paving future research.
Differences in muscle remodeling following the same regimen
used by Lee et al. (2004), Hornberger and Farrar (2004),
Duncan et al. (1998), and Yarasheski et al. (1990) could be
related to different muscles sampled in each work, the ladder
model, such as the size and number of steps (which differed
considerably amongst different the four studies), and the
number of sessions per week, load progression, and volume in
the protocols. Thus, the ladder model is a tool capable to
induce positive adaptations in muscle hypertrophy; however,

BASIC MODELS MODELING RESISTANCE TRAINING

minor modifications to the protocols may greatly affect the

results such that the functionality of the model is reduced when
muscular hypertrophy is a major endpoint, thereby reducing
the ability to study the effects of genetic manipulation or
ergogenic aids.
To monitor the variance in overload and work performed
between groups we suggest measuring venous lactate and
modifying the load appropriately. Scheffer et al. (2012)
demonstrated the effectiveness of this method to equalize the
load between groups. Additionally, we suggest researchers
using this model to induce hypertrophy modify the length of the
ladder by reducing the number of steps the animal climbs and
increasing the load to more closely mimic human strength
training. As an example Scheffer et al. (2012) employed a
hypertrophy protocol of 48 steps with 1.1 cm between steps.
Although hypertrophy was not measured, a relationship exists
between exercise-induced oxidative stress and muscle
hypertrophy (Wadley, 2013), suggesting that sets of less
repetitions may be most effective in inducing hypertrophy.
Additionally, this specific hypertrophy protocol on the ladder
may be the most appropriate for evaluating satellite cell
activation and differentiation with resistance training.
Another animal model of voluntary resistance exercise was
proposed by Klitgaard (1988) rats were trained to perform a
plantar extension in order to obtain a pellet of food (Fig. 1C).
The original protocol was performed in 2-year-old rats and
after 36 weeks of training plantaris muscle mass increased 24%.
On the other hand, utilizing the same protocol but in young rats
and for only 13 weeks we observed the plantaris muscle
hypertrophied by 13% (Zanchi et al., 2009). Our major finding
using this model was that the Atrogenes (MuRF-1 and
Atrogin-1), ubiquitin ligases involved in muscle proteolysis by
the proteasome, decreased only in the trained group,
demonstrating the ability of this model to modulate molecular
signaling. Since we did not measure the degree of muscle
protein synthesis or degradation in the isolated muscles, we
cannot speculate on the ability of this model to impact protein
turnover as a whole. There are two factors to consider when
using this model: (1) a longer training period is required for
hypertrophy to occur when compared to the synergist ablation
or the ladder model, and (2) this model uses starvation to
motivate the animals to perform the plantar extension. This
starvation period poses a major issue when studying
physiological responses as it affects both voluntary work and
nutrient status, and is also difficult to apply. Thus the
translation of this model to humans must be interpreted with
caution, although several acute studies in humans are
performed under starvation conditions (Fujita et al., 2007).
Tamaki et al. (1992) described a weight lifting exercise
model designed to induce muscle hypertrophy in the hind-limb
by loading the animal with a canvas jacket attached to the torso
and requiring the animal to perform a squat like exercise
(Fig. 1H). The main stimulus was provided by an electric
stimulator linked to the tail of the animal so a punishment
stimuli was applied and the animals performed a squat
like exercise of progressively increasing loads within a
hypertrophy range (6575% 1 Repetition Maximum, RM).
Compared with 60 min of treadmill sprints, acute squat
training resulted in an increase in plasma creatine kinase.
When sprint and squat training was carried outfor 12 weeks
at 45 days/week there was a 12% increase in the plantaris
muscle compared with control animals receiving an electric
stimulus; however, there were no significant differences
compared to the sprint training group. Although this model
contains a similar biomechanical loading and movement pattern
to human resistance training, its ability to overload the animals
and induce muscle hypertrophy is inferior to other voluntary
resistance training models, such as the ladder climb or food
motivated plantar extension proposed by Klitgaard (1988).
JOURNAL OF CELLULAR PHYSIOLOGY

Wirth et al. (2003) developed a revolutionary model where

rats were operantly conditioned to perform a squat exercise
via both reward and punishment (Fig. 1K). Food was restricted
and rats were operantly conditioned with food rewards to
enter a vertical tube, insert its head into a weighted ring (either
70 or 700 g), lift the ring until its nose interrupted an infrared
detector, and then lower the ring. Load cells measured the
external force generated, and displacement transducers
measured the vertical displacement of the ring during each
lifting and lowering movement. The apparatus and training
procedures were computer automated. Peak force, velocity,
work, and power were calculated for each movement. Rats in
both groups easily acquired the task after 1215 training
sessions conducted 5 days/week. The median peak force, work,
and power per lift for both concentric and eccentric were
greater for the 700 g group. Importantly, 8 weeks of lifting both
70 g and 700 g five sessions per week increased plantaris, soleus,
and gastrocnemius mass compared to sedentary controls;
however, dry weight and muscular protein content was not
measured, thus it is also possible these increases may have been
partly the result of edema and/or inflammation. These results
demonstrate the utility of quantitating the biomechanics of
volitional movements and suggest that the present model
can establish and maintain controlled repetitive movements
necessary for studying injury and adaptation in muscles,
tendon, and bone. Moreover, contrary to Tamaki et al. (1992)
the absolute weight of the rats was not decreased with this
training protocol, suggesting that this positive operant model
combined with histological sampling is a valid protocol to study
responses to resistance training.
Given the potential of the models described by Wirth et al.
(2003) and Klitgaard (1988), our group (Nicastro et al., 2012b)
proposed an equipment and system of resistance exercise (RE),
based on squat-type exercise for rodents, with the ability to
more precisely control the training variables proposed in
Wirth et al. (2003). In this model we developed an operant
conditioning system composed of sound, scent, light, and
feeding devices that optimized resistance exercise performance by the animal (Fig. 1L). With this system, it was not
necessary to tie the animal into the device or impose chronic
fasting or electric shock for the animal to perform the task
proposed (muscle contraction). Furthermore, it was possible
to perform muscle function tests in vivo maximal voluntary
strength capacity (MVSC) within the context of the exercise
proposed and control variables such as intensity (percent of
MVSC or percent of body weight), volume (sets and
repetitions), rest intervals between sets, and exercise session
length. Importantly, sound was the main stimulus given to the
animals as a way to optimize learning and reinforce exercise
training. Therefore, despite experimental limitations, we
believe that this RE apparatus is closer to the physiological
context observed in humans. When testing the efficacy of this
protocol to counteract the effects of 7 days of 5 mg/day
dexamethasone (a diabetogenic and proteolytic catabolic
hormone) in a common model of skeletal muscle atrophy,
we observed that training attenuated the loss of gross muscle
mass and increased plantaris mass when compared to controls.
Additionally, we observed an increase in MVSC in trained
animals, but not controls at the end of the study (Nicastro
et al., 2012a), demonstrating the efficacy of this model to
attenuate or even prevent atrophy, and as a reliable technique
to study atrophic disease.
Variables to Evaluate When Selecting a Resistance
Training Model

According to Timson (1990), when using animal models to

evaluate muscle enlargement produced by strength training
in humans, three factors must be considered: (1) Muscle

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recruitment and adaptations in fiber characteristics; (2)

magnitude of muscle enlargement. Given the effects of varying
models of voluntary and non-voluntary resistance loading
reported by our team and others, we suggest six other factors
to consider: (3) the degree of nutrition required for a positive
reward; (4) negative reward (i.e., pain). (5) Time spent
conditioning the animal to execute the exercise; (6) duration
required to obtain muscle remodeling; (7) muscle voluntary
capacity percentage; (8) resistance training under atrophic or
diseased conditions.
1. Muscle recruitment and adaptations in fiber characteristics:
With specific study questions (i.e., sarcopenia) type II
muscle fiber hypertrophy is more relevant that gross
hypertrophy in preventing the loss of muscle mass and
function; however, not all models of training are capable of
overloading all muscle fibers and thus eliciting a substantial
degree of hypertrophy in muscles comprised of predominantly Type II fibers. As an example, the plantaris is a mixed
fiber muscle and its hypertrophy through surgical ablation of
the gastrocnemius (a predominantly type II muscle) may not
be appropriate to study the reversion of sarcopenia
compared to a squat based model (Nicastro et al., 2012b).
2. Magnitude of muscle enlargement: Some exercise models are
difficult to perform and the resulting hypertrophy degree is
very poor when compared with others. For example, the
ladder climb model is capable to generate hypertrophy in
the muscles of the lower limbs of the rat; however, the
hypertrophy of the FHL vary considerably in the literature
based upon ladder length, load, and frequency (Duncan
et al., 1998; Hornberger and Farrar, 2004). In contrast,
synergist ablation results in a robust hypertrophy of the FHL
(80% hypertrophy with synergistc ablation in our group
observed by Teixeira et al., 2013, unpublished data), but not
the thigh muscles such as the rectus femoris.
3. The degree of nutrition required for a positive reward: Klitgaard
(1988) developed a model whereby a collar and an acrylic
cylinder where the rats where trained to feed through
overextension of hind-limb (plantar extension) to take the
pellet and then feed. Utilizing this model, our group (Zanchi
et al., 2009) observed it required approximately 24 h of food
restriction to motivate the animals to perform the lift in
order to obtain a food pellet. Additionally, the number of
repetitions per day was very limited (16 per day), although
we observed an increase of 13% of muscle mass (plantaris
and soleus) compared with paired feeding control group.
Thus, future investigations should consider the effects of
models that require nutrition deprivation to perform
exercise when major endpoints include robust increases
in muscle hypertrophy.
4. Negative reward: It is well known recognized that punishment is a stronger stimulus than reward to induce rodents
to perform resistance exercise (Zanchi et al., 2009).
However, sometimes this punishment stimuli is detrimental
as the appetite of the animals is reduced due to the
endocrine response involved in the fight or flight (stress)
reaction, thus impairing the muscular and molecular
adaptations to a pre-determined stimulus. For example,
Tamaki et al. (1992) demonstrated increases in gastrocnemius and plantaris mass with resistance training compared to
control groups following 12 weeks of training; however, the
resistance training group lost approximately 200 g of body
mass. Thus, punishment in this model influenced the
endocrine response and diminished the appetite of the
trained animals such that limb muscle hypertrophy was likely
compromised.
5. Time spent conditioning the animal to execute the exercise:
Through two different voluntary exercise models, we
observed that some rats are capable to learn how to
JOURNAL OF CELLULAR PHYSIOLOGY

execute a task (resistance training) very fast whereas others

are not. Therefore, every time we conducted a resistance
training protocol we selected for both control and
intervention groups only the animals capable to learn
very fast. Based on Klitgaard (1988) and Zanchi et al. (2009)
and modifying the model proposed by Wirth et al. (2003),
we developed a computerized model using both reward and
punishment stimulus to induce rodents to perform
resistance exercise (hindlimb extension; Nicastro et al.,
2012b). Based upon the results from these models, we
suggest researchers employing models of voluntary maximal
contraction spend 1416 unloaded training sessions spaced
every day over 2 weeks to condition the animals to perform
the resistance training protocol.
6. Duration required to obtain muscle remodeling: In our first
model of food rewarded plantar extension (Zanchi
et al., 2009), 3 months of training were required to increase
plantaris mass by 13%. Similarly, Tamaki et al. (1992) spent 3month to induce relative bodyweight adjusted (muscle
weight/body weight) increases in gastrocnemius and
plantaris mass of 31.4% and 17.9%, respectively, and absolute
increase of 12% in plantaris mass when compared with
controls. In comparison, utilizing electrical simulation Baar
and Esser (1999) demonstrated increases in tibialis anterior
and EDL mass of 13.9 and 14%, respectively, when
performed twice a week for 6 weeks. Further research is
required to elucidate the optimal combination of frequency
(sessions per week) and volume when designing animal
models leading to muscle hypertrophy. Despite similar
increases in absolute plantaris weight, the rats in Tamaki
et al. (1992) were trained two to three sessions per week
more than those in our study. These results are further
confounded by synergist ablation where the target muscle
(usually the plantaris) is chronically under a load every time
the rat moves (similar to a human carrying extra bodyweight, not taking part in resistance training). Thus, each
model has specific characteristics, and based upon the load
(light or heavy) and expected outcomes pilot sessions
should be conducted to determine appropriate frequencies
and study durations.
7. Muscle Voluntary Capacity Percentage: It has been well
described in the literature that intensities in the range of
7585% 1 RM are ideal to increase/hypertrophy the muscle
mass. However, utilizing some models it is almost
impossible to predict those ideal ranges. For example,
utilizing the ladder model some investigators have employed
a maximum effort test in the ladder model; however, the
ladder contains 1624 steps so the rodent is performing, in
fact, a test of local muscular endurance (i.e., a 16 RM test)
and not a true 1 RM. Predicting the maximal voluntary
power output of a rodent for a given stimulus is also difficult
and unreliable. For example, when we tested the foodreward model developed by Klitgaard (1988) the rats were
unable to perform more than eight repetitions per workout
(Zanchi et al., 2009). A major concern was whether the rat
actually exerted maximal effort, or if it was satisfied by a
reduced food intake. This issue was partially resolved in our
more recent model (Nicastro et al., 2012b) as we observed
the rats performed nearly 50 repetitions per session.
Moreover, we were able to measure force developed
through a computerized model and compare that to MVSC
to ensure the animals were working the hypertrophy range.
Thus, we believe this voluntary model allows the researcher
to more precisely manipulate volume and intensity in the
study of training induced muscular adaptations, specifically
hypertrophy and soft tissue remodeling.
8. Resistance training under atrophic or diseased conditions:
Several models of atrophy have been described in the
literature; the most commonly used mimics that of

BASIC MODELS MODELING RESISTANCE TRAINING

spaceflight (gravitational unloading) via hind limb suspension. Under this condition of atrophy, isometric-based
resistance training models capable of producing robust
hypertrophy under gravitational conditions have failed to
induce hypertrophy or counteract atrophy (Haddad
et al., 2006). It is possible that this resistance training
model may have attenuated the loss of muscle mass under a
gravitational model of atrophy (i.e., administration of
rapamyacin). On the other hand, it is well established in
the literature that dynamic resistance training plus
nutritional support (mainly proteins containing high biological value and leucine) are capable of robustly activating
protein synthesis pathways (Phillips, 2011) and counteracting unloading-induced loss of muscle mass as demonstrated by Fluckey et al. (2002). When considering the
catabolic state as generated by glucocorticoids and diabetes
mellitus, we demonstrated that resistance training is capable
of counteracting the loss of muscle mass (Nicastro
et al., 2012a). Although further research in humans is
needed to describe the hormonal milieu and muscle
activation in response to daily living activities in humans
with atrophic disease, our model provides researchers with
a greater degree of control over the variables involved in
resistance training, and can be used to study the effects of
resistance training on atrophy during conditions of
gravitational unloading and glucocorticoid catabolism.
In example of how these factors may affect investigation
outcomes, Miyazaki et al. (2011) utilized the synergistic
ablation surgery to investigate the involvement of ERK/MERK
pathways and the well-known Akt/mTOR/P70S6K pathways of
protein synthesis initiation in the muscle hypertrophy
phenomena. Using the synergist ablation model of muscle
hypertrophy, early and late periods of muscle adaption were
examined. Specifically, they measured these adaptations in a
model of form follows signaling function observing plantaris
hypertrophy weight from day 0 to day 10 on a daily basis.
Miyazaki et al. (2011) demonstrated that Akt phosphorylation
(Ser 473 or Thr 308) was not activated until days 23, whereas
P70S6K (Ser 389, Thr 421/424) and ribosomal protein (RPS6
Ser 235236) where highly phosphorylated during the entire
hypertrophy process. Of note, this delay in the classical Akt/
mTOR activation was accompanied by a rapid and prolonged
MEK 1 and 2 (Ser 217/221) activation. The same pattern was
observed in ERK 1 and 2 (Thr 221 and 224). Importantly, this
divergence utilizing a well-recognized animal muscle
hypertrophy model demonstrated parallel pathways, one
activated early and one later, that both phosphorylated the RP6
protein culminating in increased protein synthesis. In the early
pathway ribosomal kinases phosphorylated the RP6 in the Ser
235/236 residue. Following the late pathways, RPS6 was
phosphorylated in the residues Ser 240/244. This study
demonstrates the importance of matching expected outcomes
within the research question to the resistance training model
employed. If Miyazaki et al. (2011) had utilized a model
requiring a longer duration of conditioning or training to induce
muscle remodeling (i.e., the squat model) these novel
discoveries may not have been made. Thus, investigators need
to be aware of these particular attributes to resistance training
models when investigating molecular signaling pathways,
ergogenic aids, and mechanical tensions and muscle
remodeling.
Conclusion

Many important molecular findings regulating skeletal muscle

remodeling have been made through the use of diverse
experimental resistance training models. Although a number of
models are capable of inducing muscular hypertrophy,
JOURNAL OF CELLULAR PHYSIOLOGY

investigators should consider several new variables when

selecting the most appropriate model to answer the research
question. For example, investigators should be aware of the
early and late signaling pathways leading to hypertrophy, and
chose a model that activates the appropriate phase. In this same
regard, if a large degree of muscular remodeling is required to
answer a research question selecting a hypertrophy model with
a weak magnitude will result in compromised findings.
A major issue facing clinic scientists investigating the
medicinal/therapeutic effects of resistance training is that
genetic modifications in animals that are available to basic
scientists are not applicable to the general population.
Consequently, a growing emphasis is being placed on
investigating the efficacy and effectiveness of nutritional
supplements and ergogenic aids in conjunction with resistance
training. Although the discovery of new signaling pathways has
not suddenly changed or advanced the methods people use to
train, overtime these discoveries have led to more effective
training methods, especially when resistance training is used as
physiotherapy. As the ability to more accurately manipulate the
variables involved in rodent resistance training models
(volume, intensity, frequency, etc.) improve, scientists will be
better able to study the effects of distinct differences in
program design on molecular signaling and hypertrophy.
These improvements in model design and the results obtained
can then be used to improve the translation between basic
scientific discoveries, clinical practices, and the application to
human health and performance.
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