Endocrinology of Physical Activity and Sport

© Springer Nature Switzerland AG 2020

A. C. Hackney, N. W. Constantini (eds.)Endocrinology of Physical Activity and SportContemporary Endocrinologyhttps://doi.org/10.1007/978-3-030-33376-8_1

1. Methodological Considerations in Exercise Endocrinology

Anthony C. Hackney¹  , Abbie E. Smith-Ryan² and Julius E. Fink³

(1)

Department of Exercise & Sport Science, Department of Nutrition, University of North Carolina, Chapel Hill, NC, USA

(2)

Department of Exercise & Sport Science, University of North Carolina, Chapel Hill, NC, USA

(3)

Juntendo University Graduate School of Medicine, Tokyo, Japan

Anthony C. Hackney

Email: ach@email.unc.edu

Keywords

Bioanalytical proceduresHormonal responsesBiomarker data transformations-reductionScientific Method

Introduction

Over the last several decades, an increasing number of exercise science investigations have incorporated measurements of endocrine function (e.g., hormones, cytokines) into their research designs and protocols [1, 2]. This approach has allowed for a heightened level of investigation into research which examines the physiological mechanisms associated with clinical and performance-related conditions found in individuals involved in exercise training.

Some exercise science investigations, however, have not always controlled certain critical factors (e.g., time of day for blood sampling, level of chronic training, etc.) that can influence many of the hormones associated with the human endocrine system. This lack of investigative control has often resulted in the resulting research findings to be inconsistent, contradictory, and sometimes extremely difficult to interpret. This insufficient control of biological experimental factors appears to be due in part to limited knowledge by exercise science researchers in the area of clinical endocrine methodology and techniques.

Experts suggest that the factors that influence hormonal measurements, and contribute to variance in experimental outcomes, can be categorized as consisting of two potential sources: factors affecting physiological variation (i.e., affiliated with the physiological function status of the subject) and factors affecting procedural-analytical variation (i.e., determined by the investigators conducting research) [1, 3]. Regardless of the source of variance, subject, or investigator derived, if it is not controlled or accounted for appropriately, the resulting hormonal measurements obtained can be compromised and thus call into question the scientific validity of a research study.

The focus of this chapter is to provide background information for exercise science researchers on those physiological-procedural-analytical factors that can potentially affect endocrine measurements. The intent is for this material to serve as an introductory fundamental coverage on this topic in hopes of improving the quality of research in exercise endocrinology.

The field of endocrinology uses numerous abbreviations for the many of the hormones that exist. To aid those researchers unfamiliar with this lexicon, Table 1.1 lists those abbreviations for the most common hormones associated with the area of exercise science [4].

Table 1.1

The following are abbreviations commonly used for various hormones seen in exercise science and sport medicine endocrinological research (see Ref. [4])

Physiological Factors

As mentioned, factors that can influence hormonal measurements can be categorized into two broad areas: physiological and procedural-analytical. The physiological factors are those that are determined to be connected in some way to a biological function or status of the research subject (patient) at the time of the collection of the specimen (e.g., blood) to be analyzed. These are factors that can be viewed as pertaining to a variety of endogenous aspects of the subject/patient.

Sex/Gender

It appears that until the onset of puberty, there is little difference between males and females in their resting hormonal profile. Once puberty is reached though, there is increased androgenic steroid hormone production in the male, and the female starts the characteristic menstrual cycle pulsatile release of gonadotropin and sex steroid hormones [5–7]. Additionally, at puberty, resting leptin (an adipocyte cytokine; a low molecular weight protein that has endocrine-like actions on select physiological process such as the immune system [8]) levels tend to become increased in females, as compared to those in males [9]. In adulthood, the hormonal differences that begin to manifest at puberty tend to remain until females reach the postmenopausal period and males reach andropause [8, 9].

There are some sex-specific differences in the hormonal responses to exercise in males and females. These include an earlier and greater rise in testosterone and creatine kinase during exercise and up to 24 hours after exercise in males as compared to females [1, 10, 11] and a greater pre-exercise growth hormone response in females. Furthermore, the magnitude of the sex steroid hormonal response to exercise in females is influenced by the status and phase of their menstrual cycle [10, 12]. Interestingly, the menstrual cycle hormones can influence other hormones and their response to exercise (e.g., increased estradiol-β-17 → increases growth hormone levels) [10–13] (see later discussion concerning the menstrual cycle in this chapter, as well as Chap. 16 in this book). On the other hand, some hormones show little or no differences in response to exercise between the sexes (e.g., water balance hormones such as aldosterone and arginine vasopressin) [5, 10, 12].

Due to these potential differences in outcomes due to sex, the researcher should be cautious when using adult subject populations involving a mixture of males and females in their studies. To avoid confounding results, researchers need to be certain that the hormonal outcomes they are measuring are not influenced by sex/gender and stage of menstrual cycle, as will be discussed in subsequent sections of this chapter.

Age

If subjects are not matched for age and maturity level, whenever possible, variance in the outcomes can be potentially increased. For example, a prepubertal and postpubertal child (of the same gender) will not typically display the exact same hormonal exercise responses or relationships [14, 15]. This is illustrated by the well-documented increase in insulin resistance which is observed as an adolescent goes through puberty [16].

This concern should also be extended to the other end of the age spectrum. That is, a postmenopausal female or andropausal male could have drastically different hormonal responses when compared to a relative prepausal individual. For example, basal growth hormone and testosterone typically decrease with age, while cortisol and insulin resistance increase [17–19].

These types of age-related differences cannot only exist at rest, but also in response to exercise, and even after completing an exercise training program. As an illustration, a study by Brook et al. showed that the anabolic responses to resistance training are impaired within the elderly, possibly due to lower testosterone levels (young subjects = 367 ± 19; old subjects = 274 ± 19 ng•dl−1), ribosomal biogenesis (RNA:DNA ratio and c-MYC induction; young = +4 ± 2-fold change; old = +1.9 ± 1-fold change), and/or translational efficiency S6K1 phosphorylation (young = +10 ± 4-fold change; old = +4 ± 2-fold change) (see Chap. 21 in this book concerning S6K1 [mTOR substrate S6 kinase 1]) [20]. For this reason, it is important to match subjects in research studies by chronological age and/or maturation level in order to increase the homogeneity of the responses and decrease interindividual variability, obviously, that is, unless the researcher is trying to study age-related changes among groups of individuals [3].

Ethnicity and Race

A variety of different humoral constituents are known to vary between people of different races and ethnic groups [1, 3]. For example, resting parathyroid hormone levels tend to be higher in Black compared to Caucasian individuals [21]. Caucasian females tend to have higher levels of estrogens than Asian females [1, 22]. Evidence also suggests that reproductive hormone levels during gestational periods may vary greatly across several races and ethnic groups (Caucasians, Blacks, Latinos, Asians, and Indians) [22–25]. Findings of greater resting insulin and degree of insulin resistance in certain Native American tribes (e.g., Pima Indians) have also been reported; however these differences may in fact be more related to obesity issues in these individuals [26]. Testosterone levels seem also to have differences among ethnicities, with Asian- and Indian-related ethnicities showing slightly lower levels as compared to other ethnicities [27].

Hormonal responses to exercise and exercise training related to race and ethnicity have not been well studied, and the limited available findings do not suggest drastically different response outcomes beyond basal differences. Further research is certainly necessary and warranted in this area [1, 25, 26].

Body Composition: Adiposity

The level of adiposity of the body can greatly influence the release of certain cytokines by adipose tissue [3, 8, 9]. These cytokines in turn can have autocrine-, paracrine-, and endocrine-like actions and influence aspects of metabolism, reproductive, and inflammatory function [2, 3, 8, 9]. For example, increase in adipose tissue raises the expression of aromatase, triggering higher conversion rates of testosterone to estradiol, triggering a negative feedback to the pituitary gonadotropin secretion, and ultimately resulting in lower testosterone levels [28]. Additionally, several of these cytokines have been directly linked to the promotion of increased hormonal levels (e.g., increased interleukin-6 → increased cortisol) [8]. This situation becomes compounded as adiposity reaches the level of obesity and subsequently affects many hormones to a far greater degree. For example, insulin and leptin levels tend to be appreciably elevated at rest in many obese persons [29–33].

As levels of adiposity increase, the hormonal response to exercise and exercise training can change considerably from that of a normal-weight person. As an illustration, in obese persons, catecholamine and growth hormone response to exercise becomes blunted [33]. Cortisol responses to exercise seem to become elevated in some overweight-obese individuals, although isolated cases have shown cortisol responses have been shown to be blunted and reduced [32, 33]. Exercise training often allows a loss of body mass, in particular fat mass, which helps to normalize these hormones with levels observed in normal-weight people [33–37].

To ensure that varying levels of body composition of subjects will not confound hormonal outcomes, investigators need to match their subjects for adiposity as closely as possible and not just use body weight matching as criterion. Exactly how close of a match is needed is not known, but grouping normal-weight, overweight (body mass index (BMI) ≥ 25.0–< 30.0 kg/m²), and obese (BMI ≥ 30.0 kg/m²) individuals into the same subject group can most certainly complicate and add variance to some hormonal outcomes [1, 33].

Disease States

Several disease conditions such as HIV, testicular cancer, hormonal disorders (e.g., Cushing’s syndrome, Graves’ disease, etc.), infections, chronic liver or kidney diseases, type 2 diabetes, and obesity have been shown to affect hormones; e.g., lower sex hormone levels [38, 39]. Specifically, infection-type diseases may lead to testicular dysfunction, and metabolic conditions may lead to hypogonadism via increased adipose tissue and inflammation [28]. Indeed, in HIV-infected patients, lymphoma, or syphilis effects on the pituitary, can trigger or mimic apoplexy and meningeal or pituitary infection leading to fibrosis and ultimately dysfunction [40]. Increased adipose tissue often observed in obese and patients with type 2 diabetes leads to increased aromatization activity converting testosterone to estradiol, leading to a negative feedback to the pituitary gonadotropin secretion triggering hypogonadism [28]. Many individuals are unaware of these conditions when signing up for a study; therefore thorough medical screening is an important consideration.

Mental Health

Select mental health conditions and states are associated with high levels of anxiety and apprehension (e.g., posttraumatic stress disorder), which can lead to enhanced activity of the sympathetic nervous system and hypothalamic-pituitary-adrenal axis [41–43]. Subsequently, resting levels of circulating catecholamines, adrenocorticotropic hormone, β-endorphin, and cortisol can be elevated. In contrast, persons who are experiencing depression can have low arousal levels, and the abovementioned hormones could be suppressed. Moreover, depression is sometimes accompanied by low activity levels in the hypothalamic-pituitary-thyroid axis (i.e., low thyrotropin-releasing hormone, thyroid-stimulating hormone, thyroxine, and triiodothyronine) creating a euthyroid sick syndrome response [41–43]. These alterations in resting hormonal levels from such conditions can in turn result in altered hormonal responses to exercise and exercise training in individuals who have high levels of anxiety [44–46]. In some cases this can result in heightened responses (excessive) or diminished responses [44–46]. Evaluating the mental health status, via the completion of a screening questionnaire by a participant, can serve as an excellent tool to determine if a potential emotional or psychological problem exists which could confound hormonal measurements. A variety of such screening tools are available, and the reader is directed to several excellent references for overviews of this topic [47, 48]. Importantly, it is highly advisable that any such assessment be performed by a trained, qualified individual.

Menstrual Cycle

Menstrual status (eumenorrheic vs. oligomenorrheic vs. amenorrheic) and cycle phase (follicular, ovulation, luteal) in females can produce basal changes in key reproductive hormones such as estradiol-β-17, progesterone, luteinizing hormone (LH), testosterone, and follicle-stimulating hormone (FSH). These changes can be large and dramatic within select individuals. For example, the ovulatory and luteal phases result in increases in all of the aforementioned hormones above what is seen in the follicular phase (e.g., 2–10-fold greater in eumenorrheic female) [49]. These typical changes are depicted in Fig. 1.1. As noted earlier, select reproductive hormones (sex steroids) at rest can influence certain other nonreproductive hormones and nonreproductive physiological function such as estradiol-β-17 enhancing growth hormone release and thus subsequently increased lipid metabolism [11, 50, 51].

Fig. 1.1

Typical hormone changes (arbitrary scaling for concentration changes) associated with the menstrual cycle in eumenorrheic women

The menstrual status and cycle phase hormonal influences can carry over to have an impact on exercise and exercise training responses, too. Consequently, researchers may need to conduct exercise testing with females of similar menstrual status and/or in similar phases of their cycle. This precaution is also applicable to females who are using oral contraceptives, which can mimic some hormonal fluctuations similar to cycle phase changes [51, 52]. The precise impact of oral conceptive (OC) depends upon the composition of the OC used (mono-, bi-, or triphasic) and the dosage of the active estrogen and progestin agents in the pharmaceuticals.

It is also an important consideration when there is an absence of a menstrual cycle either due to amenorrhea (especially hypothalamic based) or due to pregnancy, this absence leads to changes in hormonal levels and exercise responses [1, 2, 24].

Circadian Rhythms

Over the course of a 24-h period, many hormonal levels will fluctuate and display circadian variations (see Chap. 20 of this book). In some cases these variances are due to pulse generator aspects, which is the spontaneous release of select hypothalamic hormonal releasing factors/hormones [53] within the endocrine regulatory axis. In other cases, variances are related to humoral stimuli, changes brought on by individual behavior or environmental factors, and these humoral stimuli influence hormonal release [54, 55]. Circadian hormones can display dramatic changes in levels due to their rhythm patterns, cortisol being a prime example. Morning cortisol levels are typically twice that of those found later in the day [56–58]. Table 1.2 provides some reference on the circadian pattern seen in some key hormones.

Table 1.2

Hormones that display discernable circadian patterns. The arrows indicate a relative direction for changes in concentration levels

Arrows indicate an increase (↑) or decrease (↓) in hormone concentration

These fluctuations and circadian variations need to be addressed when conducting exercise research. Studies demonstrate that the magnitude of exercise responses may not be similar at different times of the day, even if the exercise intensity and duration are held constant [1, 56]. Investigators should plan accordingly so as to more carefully control and replicate the time of day in which research evaluations are conducted and hormonal specimen collected [59, 60].

Total Versus Free Hormone Concentration

A number of hormones exist in the circulation as either in there free or total amounts forms, the latter being the sum of the free and the carrier-bound portion of the hormone. Steroid hormones are the principal example of this situation. Some investigators do not completely recognize this point and in conducting their research at times measure the wrong form of the hormone in question. A prime illustration of this is the hormone testosterone in which the free form is viewed as more biologically active.

That is, the major part of circulating testosterone binds to sex hormone-binding globulin (SHBG) and some to albumin, leaving only a small fraction of testosterone as free form. In order to be bioactive, testosterone has to be unbound, making only free and albumin-bound (weak bound easy to dissociate) available for tissue uptake. It has been debated for a long time which form of testosterone (total vs. free) is a better indicator for testosterone levels in subjects. A recent study demonstrated that even if total testosterone levels are normal, low free testosterone is associated with hypogonadal signs and symptoms. This suggests free testosterone might be a more accurate measure of androgen-related conditions as compared to total testosterone, although clinicians/researchers must make their own determination on which hormonal form they should be accessing [61].

Procedural-Analytical Factors

The second category of factors influencing hormonal measurements is made up of those factors that have procedural or analytical aspects to them. These factors are determined, selected, or in some way potentially controlled for by the investigators conducting or the participant involved with the research [1]. These factors can be viewed as exogenous relative to their influence.

Ambient Environment

When conducting research investigations, it is important to remember that excessive exposure to hot or cold ambient temperatures can stimulate the release of various hormones, e.g., those involved in water balance (aldosterone) or energy substrate mobilization (cortisol) [44, 62, 63]. Even elevated ambient relative humidity (water vapor) can induce this effect, primarily due to a compromised heat dissipation through reduced evaporative efficiency adding to the body core temperature [63]. These effects can be further augmented if hypoxemia is induced along with temperature extremes (e.g., mountain climbing), as can occur when moving to higher elevations and being exposed to greater degrees of hypoxia [64–66].

Many of the exercise and exercise training hormonal responses are tremendously impacted by environmental factors. In particular, catecholamines, growth hormone, aldosterone, arginine vasopressin, adrenocorticotropic hormone, and cortisol are all susceptible to changes in environmental conditions and show highly exacerbated responses in such varying conditions [1, 44, 62, 63].

To minimize these influences, it is critical to conduct exercise testing in controlled, standardized conditions such as in a laboratory. On the other hand, if conducting field research (where environmental standardization can be impossible), then it is important to measure/record environmental factors and convey them in any subsequent reporting of the data in the literature.

Nutrition

The nutritional status and practices of a research subject, including food composition, caloric intake, and timing of meals, can greatly impact the hormones associated with energy substrate mobilization and utilization (e.g., insulin, glucagon, epinephrine, growth hormone, insulin-like growth factor, cortisol) [1, 67, 68]. The exact nature of the effect (augmented or attenuation) depends on the interaction of the nutritional factors just mentioned and how severely the alterations are from the normal nutritional regimes of the individual [1, 33, 41].

The hormones noted above are critical during exercise to ensure that energy metabolism meets the demands of exercise. Thus, altered dietary practices and nutrition status of a subject can alter energy substrate (glycogen) storage and availability [68–70]. This in turn can cause the hormonal response to exercise to vary to some degree. For example, Galbo and associates demonstrated that the glucagon, epinephrine, growth hormone, and cortisol response to exercise were greater when a low-carbohydrate, high-fat diet is consumed (i.e., 4 days of consumption) compared to a normal mixed diet [67].

Normally in clinical settings, it is recommended that subjects be fasted prior to blood hormonal evaluations (e.g., 8 h). It is not always practical, however, for athletes to comply with such request due to their high demand for adequate caloric intake to maintain energy balance, anabolism, and muscle glycogen reserves. Therefore, a modified fasted approach may be necessary for this special population such as only a 4–6-h fast. Even with the constraints of working around an athlete’s special needs, it is still advisable that exercise investigators try to control and standardize the dietary practices of their subjects as much as possible to mitigate the effects of differing diet between subjects, and within an individual subject’s diet, if a repeated measures research design is being used [41, 67].

Nutrient Timing

The concept of nutrient timing is arguably one of the most important aspects to account for when designing a study and evaluating results [71]. The evaluation of timing food consumption has been shown to influence muscle morphology outcomes directly and indirectly by stimulating hormone secretion [72]. As coined by Dr. John Ivy, the nutrient timing system accounts for three phases: the energy phase, anabolic phase, and growth phase [71]. Additional consideration should be given to the pre-exercise phase, which can largely influence the endocrine response during and post-exercise. Although most research protocols hold diet constant, considerations for what the subject consumes before and after, ad libitum, may have substantial influences on acute and chronic adaptations, in part due to the stimulation of hormones.

Pre-exercise

Cortisol levels, which help to maintain the integrity of the immune system, are strongly influenced by glucose availability [73–75]. Additionally, acute carbohydrate intake can stimulate an increase in insulin and glucose levels, sparing muscle glycogen as well as reducing cortisol levels. Acute consumption of a glucose-electrolyte solution (GES) prior to exercise has been shown to significantly reduce cortisol levels, when compared to water. Allowing a subject to consume a carbohydrate drink before testing, independent of amount (e.g., 25 g vs. 200 g), may significantly maintain glucose and cortisol levels post-exercise, as well as stabilizing the neutrophil to lymphocyte ratio [75]. Pre-exercise vitamin consumption, or an antioxidant enhanced beverage, may protect against acute tissue damage augmenting exercise adaptations and when consumed chronically may maintain immune system markers [76].

Anabolic Phase (During Exercise)

Carbohydrate supplementation during exercise has also been associated with a blunted cortisol, growth hormone, and cytokine response while also maintaining glucose levels and insulin stability [77, 78]. There is additional evidence demonstrating reduced T cell and NK cell levels with carbohydrate feeding during exercise [77]. Acute, uncontrolled feedings should be accounted for when establishing a study design as well as potential confounders when interpreting immune function results. Protein consumption during exercise blunts protein degradation and has a sparing effect on muscle glycogen [72].

Growth Phase (Post-exercise)

Immediate post-workout fuel consumption has the potential to highly influence muscle machinery by utilizing the anabolic characteristics of insulin. Additionally, an increase in insulin post-exercise can enhance muscle glycogen resynthesis and is enhanced with a protein/carbohydrate combination [72, 79]. A carbohydrate-amino acid supplement influenced testosterone and cortisol levels 120 min after intake and exercise [80]. However, post-exercise nutrient consumption consumed later following exercise (i.e., 8–9 h) has demonstrated no hormonal influence [81]. Intake of carbohydrates + protein + vitamins post-exercise has been shown to reduce free radicals and maintain immune function. This may be a consideration for researchers evaluating exercise and immunology characteristics, as well as various aspect of overtraining.

Meal Frequency and Patterning

Meal frequency and overall caloric consumption may also influence metabolic-hormonal markers, such as C-reactive protein, fasting plasma glucose, insulin, as well as total cholesterol [82, 83]. In as much, investigators may consider questioning participants about food consumption patterns or utilize a food frequency questionnaire.

Eating Disorders

The eating disorder anorexia nervosa is a special concern relative to nutrition status due to its profound effect on the endocrine system [1, 52, 84]. Anorexics tend to have lower resting luteinizing hormone, follicle-stimulating hormone, and estradiol-β-17 levels [84]. Anorexia also affects the pituitary-thyroid-glandular axis. Specifically, the condition is associated with suppression of triiodothyronine, somewhat decreased thyroxine, elevation of reverse triiodothyronine, and, occasionally, decreases in thyroid-stimulating hormone [84]. Such a thyroidal state is referred to as the euthyroid sick syndrome and can accompany severe body weight loss [3, 52, 84]. There is also an effect on the adrenocortical axis, with higher levels of cortisol due to an increased liberation of corticotropin-releasing hormone [84]. Growth hormone is also increased, although insulin-like growth factor-1 levels (which facilitate the physiological actions of growth hormone) are suppressed in the anorexia condition [84]. Due to the psychological aspects of the anorexia nervosa (see Refs. [85, 86]), this condition could, in the context of the organization of this chapter, be also discussed with mental health issues. Thus this factor could also be considered of a biological nature and consequently has powerful effects on a multitude of endocrine measurements.

Stress-Sleep

Emotional stress and/or sleep deprivation are each known to affect certain hormones within the endocrine system. For example, emotionally distraught individuals will typically have elevated basal catecholamine, growth hormone, cortisol, and prolactin levels [1, 87–89]. Those hormones with circadian patterns (see Circadian Rhythm section; e.g., luteinizing hormone, follicle-stimulating hormone, adrenocorticotropic hormone, cortisol) can be shifted in their characteristic pattern-rhythm by disruption of sleep cycles [43, 46, 87–91].

These types of factors (i.e., stress, sleep deprivation) can also influence the hormonal response to exercise and exercise training. Investigators must attempt to control these factors whenever possible. In fact, it is advisable to have a pre-exercise questionnaire completed by a subject to monitor and evaluate the level of these factors, and if a predetermined status is not obtained, then hormonal measures and exercise testing should be rescheduled.

As a footnote to this issue, many investigations in the exercise area use college students as research subjects. Such students can have high levels of emotional stress due to their education demands (e.g., examination periods, projects being due, oral reports). Care should be taken to not utilize student subjects when there are in high emotion stress periods as a multitude of hormones can potentially have very atypical values and responses [43].

Physical Activity

The proximity in time between exercise sessions can affect the hormonal profiles of individuals [92, 93]. If inadequate amounts of time have elapsed (lack of recovery), some hormonal responses at rest, or in the subsequent exercise testing, can be attenuated and others augmented. Furthermore, the magnitude of this effect can be influenced by the exertion required of the prior exercise (e.g., high-intensity intervals require longer recovery).

If possible, researchers may require a 24-h recovery prior to a subject reporting to the laboratory for testing. However, subjects who are athletes may find it difficult to reduce their training or miss a workout session for experimental purposes in research studies. A modified approach may be necessary, such as only a 12- or 8-h recovery period, because this could somewhat prevent stress and anxiety (which as noted can affect the endocrine system) in the athlete since they would be missing less training time [1, 93–95].

A powerful influence on resting and exercise hormonal response of a subject is the exercise training status—that is, trained vs. sedentary. The more trained a subject is, typically the greater the effect on the neuroendocrine system. Many hormones show attenuated resting and submaximal exercise responses in trained individuals, although some can actually be augmented (e.g., testosterone in resistance-trained individuals) in response to submaximal and maximal exercise [2, 96–100].

Besides acute effects of physical activity on hormonal levels, chronic endurance exercise such as distance running, cycling, race walking, and triathlon has been shown to put immense stress on the endocrine system, many times resulting in the suppression of some hormones [9]. This phenomenon may be related to the overtraining syndrome (see Chap. 27 of this book). The exact mechanism of hormonal reduction following chronic strenuous endurance exercise is not elucidated yet; however an impairment of the hypothalamic-pituitary regulatory axis due to energy decreases is postulated by some researchers [101].

An extensive dialogue on the influence of exercise training on hormonal profiles at rest and in response to exercise is beyond the scope of this chapter, but the reader is directed to Refs. [2, 3] for more in-depth discussions.

Subject Posture-Position

There are changes in the plasma volume component of the blood as a subject changes position. Standing upright results in a reduction of plasma volume compared to a recumbent position [102]. These shifts in the plasma fluid are in response to gravitation effects as well as alterations in capillary filtration and osmotic pressures [102]. Large molecular size hormones, or ones bound to large weight carrier proteins, could be trapped in the vascular spaces; this means that a loss of plasma fluid would increase the concentration of these hormones (hemoconcentration). Conversely, a gain of plasma fluid would decrease the concentration of these hormones (hemodilution) [44, 103]. These adjustments in fluid volume to move in or out of the vascular space due to posture shifts typically require approximately 10–30 min [102, 103].

In exercise research situations where blood is drawn to assess hormones, it is recommended that the condition of specimen collection related to the subject’s position be controlled and reported in publications. This type of information is most certainly necessary if a postural change is occurring for a 10-min or greater duration [58, 103].

Specimen Collection

Suitable precautions must be taken in the collection and storage of blood specimens to ensure they are viable for later hormonal analysis. In clinical and exercise-related blood work, venous blood is the specimen usually utilized. If the blood specimen is being obtained by venipuncture, it is important to not have the tourniquet on the subject’s arm too long (∼1 min or longer). Greater lengths of time can result in fluid movement from the vascular bed due to increased hydrostatic pressures [103]. Once collected, the blood sample should be centrifuged at ∼ 4 °C in order to separate the plasma (collection tube contains anticoagulant) or allowed to clot (collection tube is sterile) then centrifuged for serum. If centrifugation cannot be done immediately, then the blood sample should be placed on ice, but it is more prudent to centrifuge without delay. Once separated, the plasma/serum should be aliquoted and stored at a temperature of −20 to −80 °C until later analysis. Care should be given to ensure certain plasma/serum is stored in airtight cryofreeze tubes (screw-cap type is recommended), which allow for a longer storage period. It is also advisable to split up specimens into several aliquots if multiple hormonal analyses are going to be conducted. Once a sample is thawed, it has a relatively short shelf life in a refrigerator, and repeated unthawing and refreezing cycles can degrade certain hormonal constituents and compromise the validity of the analysis [104–106]. Care should be taken to ensure that the assay procedures employed are specific for plasma or serum, as in some cases these cannot be used interchangeably in the assay (e.g., adrenocorticotropic hormone is measured in plasma). Furthermore, an examination of the research literature may be necessary to determine if one form of blood component is more popular or prevalently used in research.

In blood specimens, either plasma or serum is utilized for biochemical analysis, but some hormonal measures can also be made in urine and salivary samples. In general plasma and serum give very similar values for hormonal analytes, and seldom is one considered better than the other in blood analysis [105, 106]. Be aware, however, specific assay procedures do, in some situations, have a preferred blood fluid for analysis. Thus it is critical for the researcher to know what each hormonal assay requires as the analyte and then plan accordingly. This type of information is provided by the manufacturer of the analytical supplies-components used in the assay procedures.

With respect to urine and saliva, they are attractive as specimens to collect because of their noninvasive nature. They do, however, have certain drawbacks. Urine analysis tends to be limited primarily to steroid-based hormones, and there is usually a need to collect 24-h urine specimen. The collection of 24-h urine specimens can be a tedious and demanding process for the subject. Also, urine measurements may not always be reflective of real-time hormonal status either, as urine can sit in the bladder for hours before being voided. Saliva allows for easier sampling procedure and can reflect hormonal status in a more real-time fashion. However, saliva also primarily only allows for steroid hormonal assessments (i.e., constituents that can cross from the blood into the salivary gland) [107]. Furthermore, saliva is limited to free hormonal concentrations as the protein-bound constituents typically cannot pass through the salivary gland due to their large molecular size. Research does suggest that the blood and saliva levels of hormones can mirror each other in their relative changes, but not perfectly, as correlation coefficients of only 0.7–0.8 are typically found [1, 104, 107]. Researchers must determine if these limitations preclude the use of these biological fluids in their studies [104, 107–109].

Analytical Assays

A variety of biochemical analytical methods (i.e., assays) exist for measuring hormones in biological specimens. Chromatographic, receptor, and immunological assays are all available. Perhaps the most prevalent contemporary technique in use is immunological assays, which have variations such as chemiluminescence immunoassay (CLIA), radioimmunoassays (RIA), enzyme immunoassays (EIA), enzyme-linked immunoassays (ELISA), and electrochemiluminescence immunoassays (ECLIA) [109–111]. Each of these techniques has its strengths and weaknesses, and the discussion of each is beyond the scope of this chapter, but the reader is directed to Refs. [112–114] for more background and explanation about this subject.

Researchers should always know the particular aspects of the hormonal assay techniques they plan to use in their studies. Specifically, it is important they be aware of the precision of the assay (how accurate is it?), sensitivity of the assay (how small of a change can it detect?), and the specificity of the assay (how much cross-reactivity is there with similar looking chemical structures in the specimen?). Ideally the researcher wants the most precise, highly sensitive, and specific assay they can obtain, but cost considerations can impact decision-making in these matters. It is advisable for the researcher to report precision, sensitivity, and cross-reactivity values in publications to allow readers to determine the quality of the analytical techniques and procedures of the assays that were used. Additionally, it is desirable to report in publications the coefficient of variation (CV) within an assay and between an assay for each respective hormone measured. This will allow the reader to determine how well the analytical technical procedures were carried out [114, 115]. One step to mitigate the potential between-assay CV is to collect and analyze your biological samples in batches of specimens and not as isolated specimens on a day-by-day basis. However, caution is necessary here as batches that are too large can influence your outcome by creating end of run effects within the assay. That is, running such a large number of samples in a single batch that the precision of the technician performing the assay may be compromised (i.e., procedural fatigue), or the kinetics of the specific assay may be influenced by the length of time it takes to pipette the various components in assay (i.e., in adding the chemical reagents to the first sample tubes vs. the last tubes; too much time has transpired, resulting in different lengths of time for chemical reactions to take place within the specimen tubes) [114, 115].

Data Transformations

Before conducting statistical analysis on hormonal data measured within the assays, it may be necessary to transform the data. Two of the most common endocrine transformations usually seen in literature are (1) expressing the data as a percent change from some precondition (i.e., before exercise), basal value, and (2) conducting a logarithmic conversion of the data. The first is typically done to account for relative changes in hormonal concentrations when absolute magnitude of change may be misleading. For example, a cortisol change from 276 to 331 nmol/L is highly different from a 55–110 nmol/L (20% vs. 200%) even though the absolute magnitude is identical. A 200% increase in the hormonal concentration may have many more profound physiological effects than the smaller percentage. In the second form of transformation, logarithmic transformation is normally performed due to a large degree of variance in the subject data resulting in a nonnormal distribution. This can be due to sample size issues, variance with the analytical technique, or the physiological nature of the hormone being studied. Despite the transformation used, it is vital that the researcher report to the reader in the publication if and how the data were manipulated prior to conducting the statistical analysis (and what was the rationale for performing the transformation) [109, 116].

A third data transformation that is less frequently used is the area under-the-curve (AUC) procedure. This is carried out when there are serial specimen samples (repeated measures design) from a subject. These serial values are plotted, and then an integration of the area under the plotted responses curve is determined, thus collapsing numerous data values into one response and potentially eliminating some of the variability associated with having many hormonal measurements [117]. This approach is favored by some researchers; their rationale is the overall response of the hormone, and gland in question can be better quantified. Nonetheless, the procedure can be influenced by the number of serial samples collected to determine the response curve as well as the circadian rhythm of the hormonal release. The latter point results in the need for highly variable hormones (pulsatile) to be assessed using more frequent specimen sampling because misleading results can occur if the sampling is too infrequent [118].

Statistical Analysis

The statistical analytical procedures applied to any research study data are dictated by the design of that study. Most research in the exercise area tends to employee parametric analysis (e.g., t-test, one-way ANOVA, Pearson correlation). These analytical procedures work well with endocrine data, provided that the underlying assumptions for their use are not violated (see Ref. [119] for details). Furthermore, many North American journals prefer this form of analysis due to the robust nature of the techniques and the reduced likelihood of making a type I error (indicating findings are significant when they are in fact, not). Nevertheless, nonparametric analysis (e.g., Wilcoxon signed-rank test, Mann-Whitney U test, Friedman test) can be equally applicable for endocrine use when study designs are not excessively complex and sample sizes are relatively small [119]. It is important, however, to recognize the likelihood of increasing the occurrence of a type I error with small sample sizes. Regardless of whether parametric or nonparametric analyses are used, it is vital that the researcher report in a publication of their work what the specific statistical analysis being used is and what the rationale was for their usage [118–121].

Once assays are performed and statistical results are obtained, the researcher needs to try and understand their data in order to interpret the magnitude of treatment outcomes and physiological effects. In this interpretative process, many researchers focus intently only upon obtaining statistical significance, usually a probability level less than 0.05 (p < 0.05). Obtaining such significance is important; however, a key question that has to be addressed in the data is the issue of statistical significance vs. practical (clinical) significance for the hormonal findings. To address that question, the researcher must think about and take into account the smallest clinically important positive and negative response value levels of the effect being researched, that is, the smallest change value levels that matter. Studies can be statistically significant yet largely insignificant clinically. It is important to note that large sample sizes can produce a statistically significant result even though there might be limited or no practical importance associated with the findings [122].

To this end, effect sizes (ES) are becoming an increasingly important index used to quantify the degree of practical significance of study results (see Ref. [123] for explanation to calculating the ES statistic). Once computed, the ES statistic can be a useful indicator of the practical-clinical importance of research results because it can be operationally defined; that is, it is possible to give the observed ES ratings such as negligible-trivial, moderate, or important-very large [124]. Such ratings allow the researcher to discern the form and quantity of significance they have obtained in their study findings. In addition, the ES statistic has two advantages over traditional statistical significance testing: (a) it is independent of the size of the sample, and (b) it is a scale-free index. Thus, ES can be uniformly interpreted in different studies regardless of the sample size and the original scales of the variables being examined [123, 124].

Summary and Conclusion

To conclude, over the last several decades, exercise researchers have steadily increased the number of studies conducted which have examined the hormones and the endocrine system. Unfortunately, not all investigators working in this area of research are entirely aware of the factors that must be accounted for, and controlled, in order to ensure that valid and accurate data are obtained. This chapter reviewed some of the key physiological and procedural-analytical factors that can confound endocrine data and add variance to hormonal findings and those steps to be taken to reduce the confounding factors. Implementation of these steps can greatly aid the researcher in the interpretation and understanding of their endocrine data and in turn make their research more scientifically sound.

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A. C. Hackney, N. W. Constantini (eds.)Endocrinology of Physical Activity and SportContemporary Endocrinologyhttps://doi.org/10.1007/978-3-030-33376-8_2

2. Endogenous Opiates and Exercise-Related Hypoalgesia

Allan H. Goldfarb¹  , Robert R. Kraemer²   and Brandon A. Baiamonte³  

(1)

University of North Carolina Greensboro, Department of Kinesiology, Greensboro, NC, USA

(2)

Southeastern Louisiana University, Department of Kinesiology and Health Studies, Hammond, LA, USA

(3)

Southeastern Louisiana University, Department of Psychology, Hammond, LA, USA

Allan H. Goldfarb (Corresponding author)