The testes secrete testosterone at a rate which varies in a circadian way and exerts a powerful anabolic impact on muscles (32). Recent evidence suggests that the amount of exercise time and the level of hormones, especially regarding the circadian rhythm, have some effects on physical and psychological performance (18,31). Currently, there are many unanswered questions about the responses of both endocrine hormone secretion mechanisms and salivary testosterone to intensive resistance exercise and their subsequent effects on the waking circadian scale in men, particularly in a population of young recreational lifters. Most of these questions are in relation to the following variables: the time of exercise and sampling, the variety, duration, and intensity of exercise, and the basic fitness level of the subjects, followed by the circadian rhythm (20). Investigations on the endocrine hormone levels with anabolic characteristics could be performed as a clinical method for analyzing and monitoring the physical and psychological attributes of the athletes. Also, it would inform coaches, athletes, and strength researchers in prescribing resistance exercise workouts. Therefore, the role of testosterone as an anabolic hormone must be considered.
It is generally accepted that testosterone is responsive to physical exercises and other forms of stress. Testosterone is an anabolic hormone, which promotes protein synthesis and has a major role in the growth and preservation of the muscles and tissues (2). The resulting effects on skeletal muscle anabolism are often due to a spike in the rate of testosterone during the healing stages (27). Until now, a large number of studies have used blood samples to assess the levels of hormones (7,11,12,15). Some of these studies (7,11,12) have shown how resistance exercise can create fluctuations in the levels of cortisol and testosterone. However, there is a lack of information that specially addresses the effects of intensive resistance exercise on the circadian concentrations of salivary testosterone in young male recreational lifters.
Examining testosterone levels in saliva is a convenient, noninvasive way to determine the concentrations of testosterone in the body. Previous studies (6,8,16,23) have indicated that salivary testosterone levels provide a reliable reflection of gonad function, and in particular, the circadian rhythm of the hormone, when frequent sampling is performed (5,23). A circadian rhythm has also been observed in salivary testosterone, with lower concentrations detected in the afternoon than in the morning (8,22,33). Previous research has clarified the effect of an intensive resistance exercise session performed in the early morning on the circadian rhythm of salivary testosterone in body builders (23); however, it is important to establish an understanding of the circadian variation, which could be further enhanced by the implementation of a morning resistance exercise routine (11).
The practical question is whether athletes are better off undertaking their resistance exercise in the morning or afternoon. We determined whether any significant differences in hormonal response were likely between the exercise training in the morning and in the afternoon.
As this study is essentially a variation of a similar study performed by Kraemer et al. (23), in which testing was performed solely in the morning, we will compare our results, which were based on exercise performed in the afternoon and with a slight modification in the intensity, with the findings of the aforementioned study.
We hypothesized that the circadian rhythm of testosterone concentrations is under circadian patterns, using the modified protocol (increasing the intensity), which was reported by Kraemer et al. (23), and also that altering the time of exercise from early morning to the afternoon would not change the outcome. Therefore, the purpose of this investigation was to determine the effects of intensive resistance exercise performed in the afternoon on circadian salivary testosterone concentrations among young male recreational lifters.
Experimental Approach to the Problem
In this study, a parallel group design was used to investigate the effects of intensive resistance exercise (as the independent variable) on circadian salivary testosterone concentrations (as the dependent variable) among young male recreational lifters. Subjects were either exposed to a series of resistance exercise sessions or functioned as a control group, in which they performed no exercise. The salivary testosterone concentrations of control and resistance exercise groups were examined on 2 occasions 3 weeks apart. Unstimulated saliva samples were collected at various time intervals (from 0600 to 2200 hours) to establish a measure of the circadian rhythm of testosterone secretion. Because of the literature that believes that the resistance training athletes have higher performance in the afternoon (14), this study was required to focus on different angles to investigate the variables of time and using higher intensity in the exercise session.
Twenty healthy young, recreational lifter men (age, 18.0 ± 1.3 years; body mass, 75.2 ± 3.2 kg; height, 1.79 ± 0.03 m; body fat %, 8.1 ± 1.9 [mean ± SD]) who had 2 years of experience in weightlifting participated in the study. Subjects were randomly selected to be part of either the resistance exercise (n = 10) or control (n = 10) group. All subjects were active members of weightlifting gyms in the city of Isfahan, Iran, when the study was conducted in April of 2012.
Before undergoing their physical examinations by the physician, all subjects filled out a questionnaire (23), and those who had any chronic medical condition, which could create an unnecessary risk during their exercise testing, were excluded from the study.
The resistance exercise group performed regular exercise sessions (3 times per week at 1605 hours) for 3 weeks, and the control group completed all of the required tests with the absence of physical exercises. The subjects included in this research did not consume dietary supplements in the form of carbohydrates, proteins (combined protein and carbohydrate intake impacts androgen receptors and testosterone concentrations (35)), or amino acids (amino acids affect hormonal responses (8)), nor were any of them taking anabolic steroids either before or while participating in this research.
The subjects' schedule of daily activity and diets were carefully evaluated by a dietician 2 days beforehand and during implementation of the experiment, until the final sample of saliva had been collected. The dietician instructed them on their food intake in 3–5 daily meals, which contained an approximate fat percentage of 30%, carbohydrate percentage of 50%, and protein percentage of 20%, as this combination has no demonstrable effect on the circadian rhythm of testosterone concentrations (23,31,37). A diet containing more than 44% fat is known to influence the level of testosterone (8,37).
All test subjects were given instruction on how to remain properly hydrated to avoid the possibility of hypohydration impacting their performance during the study (21) (the subjects' water consumption was recorded and found to be 3.8 ± 0.2 L daily).
The subjects were prohibited from consuming drinks containing alcohol, caffeine, or any other stimulants. They were also asked not to engage in sexual or other strenuous physical activity within 24 hours of the days in which saliva collection was to occur. The recruited subjects were not involved in other physical exercises, and both groups were advised to continue with a normal sleep pattern of approximately 8 hours per night for the duration of the experimental study period.
Ethical approval for the study was obtained from the University of Isfahan, Faculty of Physical Education and Sport Sciences, in Iran. The subjects were given a clear explanation of the objectives of the study, as well as the potential risks involved, and consent forms (parental consent forms for those younger than 18 years) were obtained for all subjects.
A modified protocol, introduced by Kraemer et al. (23) (Table 1), was used in this study. Exercises 3–8 were performed with proper machines (TechnoGym Equipments, Bracknell, United Kingdom), and the other exercise (except sit-ups) was performed with an Olympic-style barbell (Iron Grip Barbell Company, Santa Ana, CA, USA).
Replication of the Study
In this study, the intensity and time of the exercise was adapted and altered from Kraemer et al. (23). After 10–15 minutes of general warm-up exercises and stretching, the resistance exercise group subjects started the testing session at 1605 hours. They performed 3 sets of each exercise, with 6–7 repetitions and 2 minutes of rest in between each set as the exercise protocol, with a daily exercise session duration of around 1 hour and 30 minutes. The experimental process took place over a period of 3 weeks. It should be noted that in each session, the heart rate was found to be more than 180 b·min−1. For the purposes of monitoring the intensity of the exercise, the heart rates of 7 random athletes were determined based on Karvonen's method of Heart Rate Calculating (19). The environmental factors, such as the noise level (82 dB), temperature (19.0 ± 1.0° C), humidity (40–50%), and comfort, were strictly controlled. The subjects were permitted to drink 1.0 ± 0.2 L of cool water (12° C) during their exercise sessions.
Assessment of the Body Composition
Anthropometric characteristics, including height, weight, and body mass, were measured by the same investigator using standard procedure before the beginning of the study for all participants. For weight, a physician's beam digital scale (±0.10 kg), with the participants barefoot and without heavy clothing, was used, followed by a barefoot measurement using a height rod (±0.005 m) (SECA700; Chino, CA, USA). The body mass index was calculated as weight in kilograms divided by squared height in meters, and the body composition was assessed by means of a Lange sector for the cutaneous fold, which was based on the Jackson and Pollock protocol (17). A Lange skinfold caliper was used to determine the fat in skinfolds of the triceps, abdomen, and upper iliac areas of the test subjects. This process was repeated 3 times to validate the accuracy (3), and the thickness of skinfolds was used to evaluate the percentage of body fat (25).
One Repetition Maximum Measurement
Once the resistance exercise group had a full understanding of the equipment and was taught the necessary techniques by the Certified Strength and Conditioning Specialist (CSCS), they participated in the 1 repetition maximum (1RM) test. Determination of 1RM was done for bench press, leg press, and shoulder press. The participants initially performed 2 warm-up sets of 2–5 repetitions. The weight for these was set at approximately 50 and 80% of their perceived 1RM, respectively. These were followed by 3–4 sets of steadily increasing weight, which included rest intervals of 3–5 minutes, until a 1RM weight was established for each individual. The same investigator was used to monitor all of the 1RM tests, ensuring that all participants performed the exercises accurately using a complete range of motion (22).
The unstimulated saliva sampling (2 ml) pretest was performed on a day when no exercise session took place. The posttest sampling was done 3 weeks later, while the resistance exercise group did the exercise and the control group refrained from physical exertion. Samples were collected from both groups in 10 separate intervals, beginning at 0600 hours and repeated every 2 hours until 1600 hours; further samples were collected at 1730, 1800, 2000, and 2200 hours. The samples were obtained while the subjects were leaning forward in a seated position and collected by means of the passive drool method. They were asked to wash their mouths with water, and after waiting for 1 minute, deposit the unstimulated saliva (2 ml) into the receptacles (9,23). To avoid the circadian influences on their performance, sampling was conducted at the same time during the day (9,29). The subjects were asked to avoid any oral contact with objects such as floss or toothbrushes before and during the testing session (23).
The process of sample collection was monitored carefully, and the specimens were immediately transferred to the professional medical and pathology laboratory and stored at −80° C (1). To avoid interassay variation, the collected samples were all examined in the same assessment. RADIM kit (SEAC Company, Pomezia, Italy) and the quantitative ELISA protocol (ELISA Technologies, Inc., Gainesville, FL, USA) were used to determine the concentrations of testosterone. All of the samples for each subject were analyzed on the same micro plate (23).
For quality control, a few subjects were randomly selected from both groups and the experiments were repeated for a second time, and the results were 99% consistent with the original findings.
The sample size for the study was calculated using G power software, and all of the variables were subjected to the normality test. The result revealed that all variables were distributed normally to determine the mean values and SDs, and descriptive statistics were used in reporting the data. The saliva testosterone concentrations were analyzed at 10 points throughout the day (from 0600 until 2200 hours).
Regarding the statistical method, the 2-way repeated-measure analysis of variance (ANOVA) after Bonferroni post hoc test was used for within-group comparison, and all related assumptions, including the normal distribution of dependent variables and sphericity (homogeneity of variance and covariance), were met. Using Pearson's product-moment correlation coefficient, the linearity and correlations of the dependent variable among all of the 3 or more repeated measures were equal. A retest correlation was used to quantify the reliability of the correlation, which was 0.94, and represents a very high level of reliability.
The separate resistance exercise and control groups' circadian rhythm data were analyzed using repeated-measure ANOVA methods. Additionally, 2-way repeated-measure ANOVA analysis was performed, including 2 between factors (exercise and control) and 2 within factors (pre- and posttest) at the 10 sampling time periods (0600, 0800, 1000, 1200, 1400, 1600, 1730, 1800, 2000, and 2200 hours) to evaluate testosterone changes. SPSS version 19 (SPSS, Inc., Chicago, IL, USA) was used for analyzing the data. The differences for all analyses were considered significant if p ≤ 0.05.
With reference to Figure 1, one can observe the activity of testosterone levels in the condition of normal daily rest of the 2 different groups, namely exercise and control, based on the outcome of salivary samplings taken every 2 hours. The results obtained in the control group showed that peak salivary testosterone levels (607.62 ± 19.76 pmol/L) occurred in early morning (0600 hours), with a corresponding reduction throughout the day until an evening nadir (2200 hours) (323.18 ± 4.914 pmol/L). Similarly, in the RE group, the testosterone level within saliva (611.78 ± 16.406 pmol/L) was highest in early morning (0600 hours), with a gradual reduction throughout the day until an evening nadir (2200 hours) (321.88 ± 5.018 pmol/L). The data showed that the concentrations of salivary testosterone among our subjects were in the normal range reported by Teo et al. (33) (normal range, 693 ± 243 pmol/L in the early morning and 451 ± 173 pmol/L in the evening), and it was also consistent with the salivary testosterone concentrations reported in similar research performed by Kraemer et al. (23).
Both the control and resistance exercise groups had their salivary testosterone concentrations examined on 2 separate occasions 3 weeks apart. The resistance exercise group performed 3 weeks of prescribed resistance exercise and had their circadian rhythm of salivary testosterone concentrations examined to measure what difference could be observed after 3 weeks of exercise training. On the first occasion, this was done on a rest day (on which they performed no exercise), whereas on the second occasion, 3 weeks later; the measurement was taken on a day when they performed the resistance exercise session at 1600 hours. As expected, for the control group, there was no change in the circadian pattern of salivary testosterone concentrations after 3 weeks without exercise (a within-group comparison) (F = 21.9, p = 0.164). For the resistance exercise group, there was a temporary rise in salivary testosterone immediately after the training session at 1730 hours (F = 458.665, p = 0.001), but no difference at any other time point compared with the salivary testosterone concentrations on the rest day (a within group comparison) (F = 37.4, p = 0.216). Subsequent escalation of testosterone levels was also observed in the hours after the exercise, as shown in Figure 2. The intra- and interassay variance for testosterone was recorded at 2.3–6.1% and 7.7–8.1%, respectively.
With reference to this study, resting and exercise conditions exhibited no apparent differences, despite the fact that the previous time period was actually much higher than the ensuing one. However, there was a significant group × time during the workout interaction for the testosterone levels (F = 11.965, p ≤ 0.05). Bonferroni post hoc analyses indicated that only the men who performed resistance exercise significantly increased salivary testosterone, from 371.28 ± 7.228 to 513.76 ± 20.332 pmol/L, respectively (p = 0.001) (Figure 2), but that the change was temporary, and appeared not to have any lasting effects on the circadian levels of testosterone. Therefore, the comparison in the mean of testosterone secretion in pre- and posttest between resistance exercise and control groups showed that intensive resistance exercise had no significant effect on circadian secretion of salivary testosterone in 16 hours of waking time (Figure 2).
In 2001, Kraemer investigated the effect of resistance exercise in the early morning on the circadian rhythm of testosterone concentrations, and the result showed that the circadian rhythm did not change significantly (23). However, a couple of key points set this study apart from our own. First, the time when exercise training was undertaken was moved to the afternoon rather than the morning. The reason for this decision was that the afternoon is typically the time during which most lifters perform their routines (9). Second, the previous research (23) was done without a control group with which to compare results. In this study, however, a control group who did not train was used to show the differences more clearly in comparison with the resistance exercise group. Third, the previous research was done with 10 repetitions (performed at roughly 75% 1RM) for each set of each exercise. Here, we have increased the intensity (to approximately 85% 1RM) by decreasing the number of repetitions (from 10 to 6–7 repetitions) without changing the duration of rest between sets. The reason for this decision was to evoke a larger effect on hormonal responses in comparison with Kraemer's study (23).
What has not been answered yet is whether intensive resistance exercise in the afternoon has different effects on the circadian rhythm of testosterone concentrations throughout a complete day in comparison with exercise strictly in the morning. The main finding of this study was that an intense period of intensive resistance-oriented sport activities provides a nominal temporary effect upon the testosterone circadian rhythm that diminishes an hour after the workout, after which testosterone levels return to normal. The findings obtained through both resistance exercise and control groups obviously manifest a circadian pattern in which there is a presence of higher concentrations during the morning and lower concentrations during the evening, and it is under circadian patterns. These results are in line with previous studies (9,15,22,23).
This rhythm was consistent with the hypothesized rhythm for the secretion of testosterone. The measurements taken on both occasions 3 weeks apart resulted in levels, which were within a normal range as reported by Teo et al. (33), and these data were in line with the results of Kraemer et al. (23), providing further evidence of stability in the daily pattern of salivary testosterone concentrations during the waking hours.
The results of the sampling at various times over the course of a day showed that there was a remarkable increase in the level of testosterone immediately after the intensive resistance exercise at 1730 hours, and this seems to imply an acute alteration rather than a change in the circadian rhythm of testosterone secretion in response to intensive resistance exercise. The research performed by Kraemer et al. (23) likewise showed a significant increase in salivary testosterone concentrations immediately after the resistance exercise, but given the time frame in which the research was conducted, this increase was observed in the early morning, with no corresponding data regarding the afternoon. The rhythm returned to its normal level a few hours after concluding the exercise. However, the precise physiological mechanism inducing the increase in testosterone resulting from exercise remains unclear. In resting conditions, the testes were primarily under the influence of the gonadotropins (luteinizing hormone and follicle-stimulating hormone). However, no relationship has been found between the levels of the gonadotropins and the increase of testosterone regarding exercise (30,31). Meanwhile, gonadotropins work as regulators and based on the results of this study, intensive exercise (in the afternoon or morning) may not affect their performance. An increase in the level of free testosterone due to alterations in the binding affinity of the sex hormone binding globulin has also been discounted by Fahrner (11). It has been suggested that the increase in testosterone is due to a reduced metabolic clearance rate, and alternatively, Tsigos et al. (34) postulated that the increase may be mediated by sympathetic stimulation of the testes through the adrenal gland.
The results of several studies (2,4,6,8,14,28) indicate that the way in which exercise is performed, with respect to the following variables, is critical in hormonal responses: intensity and duration, type of the exercise; fitness level and gender of the subjects; rest intervals between the exercises, and the conditions under which the recovery of the muscles takes place. Certain factors, such as sleep or waking periods, nutrition, meal time, physical exercises, hormones, and stress, can influence and change the pattern of the circadian rhythm (10,27,28). Intensive resistance exercise without sufficient rest intervals and the stress caused by competitive sports can result in changes in the physiological, immunological, psychological, and functional status of the athletes in the long term (12,15,16). Physical exercises affect the hormonal responses, so considering the pattern of the circadian rhythm might lead to achieving the ideal metabolism and allow us to maximize the effect of resistance exercise on the skeletal muscles (30,38). It has been well established that intensive resistance exercise is able to dramatically boost the serum levels of testosterone (24), but the results of this study showed that this effect is temporary, and after each session of exercise will return to the normal level. It should be noted that free testosterone is rigidly controlled (e.g., by mechanisms of nitric oxide and blood flow), and these mechanisms are important factors in terms of the body's response to resistance exercise and the concentrations of testosterone (23).
Utilization of salivary testosterone proved to be trustworthy in comparison with the blood serum and accurately reflected the circadian rhythms (16), especially in frequent sampling (26).
In a report by Beaven et al. (4), it was shown that the increase in serum testosterone coincides with an increase in salivary testosterone for the first hour. Likewise, in another study, Vittek et al. (36) investigated the relation between serum and salivary testosterone. The results showed that both free and total testosterone were highly correlated (r = 0.97 and r = 0.70–0.87, respectively) and denoted that they are statistically significant. These data, in combination with the results of the present research, can be deemed as a good indication that salivary testosterone is indicative of fluctuations in free testosterone (12,22).
It is plausible that after a period of exercise, regulatory components are consistently involved due to the quick transition experienced in salivary testosterone levels and the normal circadian rhythm (13,15,16,28,33).
In the previous research by Kreamer et al. (23), they referred to their exercise protocol as a heavy protocol, but their results showed that it did not significantly affect the circadian concentrations of salivary testosterone. Therefore, we decided to make the protocol in our study more intensive by decreasing the number of repetition to 6–7 and increasing the weight, although we did not change the rest time between the sets. But, regarding the results of Kraemer et al., we hypothesized that the circadian rhythm of testosterone concentrations, using the modified protocol (increasing the intensity), which was reported by Kraemer et al. (23), and also that altering the time of exercise from early morning to the afternoon would not change the outcome in comparison with Kraemer et al. (23) research. This hypothesis was based on an individual period of resultantly intensive exercise within the length of the afternoon, which could account for the slight alterations within the circadian rhythm of waking hours. Therefore, the present hypothesis was built upon previously done studies within which long sampling times, i.e., 6–8 hours, have been applied (8). Such data denote that homeostatic processes become swiftly involved in bringing back the concentrations level of serum testosterone to usual circadian quantities (23). The findings of this study provide further support for such a conclusion, and illustrate a tight adjustment of the concentration levels of salivary testosterone subsequent to physical stress.
Kraemer et al. (23) showed that heavy resistance exercise has no significant effect on the circadian rhythm of salivary testosterone among male body builders, and that it may instead be the circadian variation, which could be enhanced through a repeated morning resistance exercise routine. Changing the time of exercise (from early morning to the afternoon) and increasing the intensity of the exercise in this study did not significantly affect the circadian rhythm of testosterone concentrations, and it had merely a temporary effect immediately after the exercise. Based on these data, it can be said that the signals for androgen receptors were directly linked to the time in which exercise training was performed, and that the decrease was most likely related to not only circadian values but also the increased activity of the exercise group, as we did not see the same pattern within the control group.
According to the aforementioned information, it seems that the results of this study, regarding testosterone, are in agreement with the findings of most of the previously published studies, especially with Kraemer et al. (23), which was only based on morning exercise training. These studies both showed that resistance exercise in the morning or afternoon did not substantially affect the circadian rhythm of testosterone concentrations and that free testosterone seems to be regulated by the body's homeostatic systems.
The evaluation and measurement of testosterone in the saliva can be regarded as a highly practical and helpful method in primary research studies and clinical environments. The results of this study showed that intensive resistance exercise has no noteworthy effect on circadian secretion of salivary testosterone throughout the 16 waking hours. Therefore, this information would be useful for athletes, coaches, and researchers, and athletes can undertake resistance exercise in the morning or afternoon with the knowledge that both will result in similar testosterone responses. It seems that there are other potential factors, such as diet, which can affect the circadian secretion of salivary testosterone. It is suggested for further researchers to investigate the circadian secretion of salivary testosterone in response to the concurrent effects of aerobic and resistance exercise.
The authors specially thank the University Putra Malaysia, Department of Sport Sciences, and University of Isfahan (Iran) Departments of Exercise Physiology. The authors also thank all the subjects who helped them in this project. The authors express their special thanks to Prof. David Pyne for reviewing the article despite his very busy schedule. Last but not least, their sincere gratitude to Matthew Ballard who helped them a lot in finalizing this text within the limited time frame. The results of this study do not constitute endorsement by the authors or the National Strength and Conditioning Association.
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