Hormonal Changes in High-Level Aerobic Male Athletes during a Sports Season

im of this study was to determine the possible changes in plasma of several hormones such as Luteinizing Hormone, Testosterone, Cortisol and Insulin in endurance runners during the sports season. Twenty-one high-level male endurance runners (22 3.2 years, 1.77 0.05 m) participated in the study. Basal plasma hormones were measured at four moments during the season (initial, 3, 6 and 9 months), and were analyzed using ELISA (enzyme-linked immunosorbent assay). Testosterone and Luteinizing Hormone (LH) su ered very signi cant decreases (p<0.01) at 3 months compared with the beginning and an increase (p<0.05) at 6 and 9 months compared with 3 months. Insulin level was signi cantly lower (p<0.05) at 3, 6 and 9 months compared with the initial test. Insulin and cortisol were associated inversely (r=0.363; = 0.577;p=0.017) and positively (r=0.202; =0.310;p=0.043), respectively, with the amount of km per week performed by the runners. There was a signi cant association between km covered at a higher intensity than the anaerobic threshold and I (r=0.580; = 0.442;p=0.000). Our ndings indicate that testosterone, LH and insulin were more sensitive to changes in training volume and intensity than cortisol in high-level endurance runners. Basal testosterone and LH concentrations decrease in athletes who perform a high volume of aerobic km in situations of low energy availability. Keywords:hormones; LH; testosterone; cortisol; insulin; athletes 1. Introduction Endurance athletes modulate the volume and intensity of their constant training throughout the sports season in order to produce adaptations and

volume and intensity than cortisol in high-level endurance runners. Basal testosterone and LH concentrations decrease in athletes who perform a high volume of aerobic km in situations of low energy availability. Keywords:hormones; LH; testosterone; cortisol; insulin; athletes 1. Introduction Endurance athletes modulate the volume and intensity of their constant training throughout the sports season in order to produce adaptations and achieve their best performance in previously established competitive periods [1]. This exercise causes stress in the organism that induces important changes in the endocrine system to recover the initial homeostasis [2]. Cortisol (C) is the main glucocorticoid of the organism, its secretion is produced in the adrenal glands and it is controlled through a negative feedback mechanism by the hypothalamus– pituitary–adrenal axis [3]. It is a hormone modulated by circadian rhythms, but factors such as mental stress, dehydration or food can alter its production [4]. In endurance activities, its blood values increase, as its catabolic function contributes to maintaining adequate energy levels through protein degradation, hydrolysis of triglycerides and even adding additional energy from carbohydrates through gluconeogenesis in the liver [5]. At the end of physical activity, the concentrations of this hormone begin to decrease, and it may take up to 48 h to recover its basal values after maximum e ort [6]. Int. J. Environ. Res. Public Health2020,17, 5833; doi:10.3390 /ijerph17165833 /journal/ijerph

Int. J. Environ. Res. Public Health2020,17, 5833 2 of 12 Testosterone (T) is an anabolic hormone that participates in multiple physiological functions, intervenes in muscle protein synthesis, stimulates bone remodeling and erythropoiesis [7] and regulates the function of lactate transporter proteins thus promoting lactate oxidation as a fuel during exercise [8]. T is secreted by the Leydig cells in the testicles and its concentration in blood is controlled by the hypothalamic–pituitary–testicular (HPT) axis [9]. It has been reported that athletes who undergo long-term continuous training may have reduced levels of chronic basal T, status de ned as “Exercise-Hipogonadal Male Condition” [10]. Previous studies have shown that its low levels would be caused by the negative relationship between C and T [11,12]. Hackney, Szczepanowska and Viru have hypothesized whether this inhibition would be caused by a dysfunction of the axis at the peripheral level, through direct inhibition of the Leydig cells in the testicles, or at the central level, reducing the release of Luteinizing Hormone (LH) in the pituitary that would a ect the production of T at the testicular level [13]. LH is secreted in the anterior pituitary gland. LH is part of a pathway comprising the hypothalamus, pituitary gland, and gonads [14]. The release of LH is stimulated by gonadotropin-releasing hormone (GnRH) [15]. After acute physical exercise, LH usually decreases [16,17]. During the training phases, a reduction in LH secretion was found in runners [18]. The T/C ratio is a variable that relates to the anabolic/catabolic balance in athletes and is widely used for monitoring and evaluating the body's response to chronic exercise-induced stress [19]. Authors such as Meeusen et al. [20] think that the ratio cannot be used as a means of control since they reported studies where decreases of 30% of the ratio did not always worsen the athletes' performance. Another important hormone involved during physical exercise is insulin (I), related to energy balance and blood glucose control [21]. Horton, Grunwald, Lavely and Donahoo reported a decrease in plasma I concentration during exercise, followed by an increase during the hours after exercise to favor glycogen repletion, a

decreases of 30% of the ratio did not always worsen the athletes' performance. Another important hormone involved during physical exercise is insulin (I), related to energy balance and blood glucose control [21]. Horton, Grunwald, Lavely and Donahoo reported a decrease in plasma I concentration during exercise, followed by an increase during the hours after exercise to favor glycogen repletion, a decrease in carbohydrate oxidation, and an increase in fat oxidation [22]. Aerobic endurance athletes have lower baseline values and higher insulin sensitivity than sedentary subjects to support fatty acid oxidation [23]. In summary, hormones have been de ned as important mediators in the body's response and adaptations to exercise-induced stress. Their acute responses to di erent stimuli and their modi cations over short periods have been extensively investigated [24,25]. However, few studies have shown hormonal changes in high-level endurance runners during a sports season, so the aim of this study was to determine the baseline values of T, C, LH and I and their changes throughout a sports season where training loads are modulated to obtain several peaks of performance. 2. Materials and Methods 2.1. Participants The athletes were studied every three months at four moments during an athletic season. The measures were made during the rst week of October, January, April and July. Athletes were informed of the purpose of the study and signed an informed consent form prior to enrolment. A code was assigned to each participant for the collection and treatment of the samples in order to maintain their anonymity. This research was carried out under the Helsinki Declaration ethical guidelines, updated at the World Medical Assembly in Fortaleza (Brazil) in 2013 for research with human subjects, and the Ethics Committee of the University of Extremadura approved the protocol (52/2012). Twenty-one high-level aerobic male runners (22 3.2 years, 1.77 0.05 m) participated in the present survey, all of them were living in the area of Caceres (Spain), at a latitude of 39 28' 35.36” N. Each athlete had at least ve years of training experience, and all of them were participants in national and international tournaments (1500

the protocol (52/2012). Twenty-one high-level aerobic male runners (22 3.2 years, 1.77 0.05 m) participated in the present survey, all of them were living in the area of Caceres (Spain), at a latitude of 39 28' 35.36” N. Each athlete had at least ve years of training experience, and all of them were participants in national and international tournaments (1500 and 5000 m race modalities). All subjects were required to have a stable body weight throughout the sports season (no weight changes>3%). Signi cant changes in fat mass and fat-free mass (FFM) are associated with circulating T concentrations due to their role in energy metabolism and adipogenesis [26]. The participants did not take regular medication,

Int. J. Environ. Res. Public Health2020,17, 5833 3 of 12 anti-in ammatory medications or nutritional supplementation during the two weeks prior to the measurements. None of the subjects had taken hormonal medication in the previous year or during the study since any high-level athlete is obliged to conform to drug testing in competition or out of competition. 2.2. Nutritional Evaluation All participants were instructed to complete a 3-day diet record, including one weekend day and two weekdays, on the provided nutritional questionnaire; each participant weighed and indicated the amount in grams of each food consumed. The athletes' dietary intakes were obtained using a food composition table [27]. 2.3. Anthropometrics Measurements Subjects reported to the laboratory after an overnight fast and had to abstain from hard training and/or competition for at least 72 h before testing. The participants' morphological characteristics were measured in the morning and always at the same time (09:00 a.m.). Body height was measured to the nearest 0.1 cm using a wall-mounted stadiometer (Seca©, Hamburg, Germany), and body weight was measured to the nearest 0.01 kg using calibrated electronic digital scales, (Seca©, Hamburg, Germany) in barefoot conditions. Fat mass and fat-free mass content was estimated from the sum of 6 skinfolds ( P 6) (abdominal, suprailiac, tricipital and subscapularis, thigh and calf). Skinfold thicknesses were measured with a Harpenden caliper (Holtain Skinfold Caliper, Crosswell, UK) and converted to % of body fat using the equations of Jackson and Pollock [28]. All measurements were made by the same operator, accredited in kinanthropometric techniques (level 1), in accordance with the International Society for the Advancement of Kinanthropometry (ISAK) recommendations [29]. 2.4. Exercise Test until Exhaustion A running test on a treadmill (Powerjoc, UK) equipped with a gas analyzer (Metamax, Cortex Biophysik Gmbh, Germany) and a Polar pulsometer (Polar Vantage M, Norway) was used to evaluate the maximum oxygen uptake (VO2max). All the tests were performed between 10 and 12 a.m. Exercise test consisted of a 10 min warm-up at 10 km/h followed by incremental runs until voluntary exhaustion, starting at 10 km/h and increasing it by 1 km/h every 400

Biophysik Gmbh, Germany) and a Polar pulsometer (Polar Vantage M, Norway) was used to evaluate the maximum oxygen uptake (VO2max). All the tests were performed between 10 and 12 a.m. Exercise test consisted of a 10 min warm-up at 10 km/h followed by incremental runs until voluntary exhaustion, starting at 10 km/h and increasing it by 1 km/h every 400 m, with a stable slope of 1%. During the incremental test, VO2max was determined according to the following criteria: the respiratory exchange ratio (RER) had to exceed 1; stabilization in oxygen uptake (VO2) together with an increment in carbon dioxide (CO2) elimination and in the ventilatory volume (VE), induced by the increases in the test velocity. After recording the test data, the ventilatory thresholds were determined according to the three-phase model to monitor training [30]. The data were obtained at the aerobic threshold (VT1) and the anaerobic threshold (VT2) to determine training load intensity. 2.5. Training Characteristics Figure four-week adaptation before the initial measurement (October), where they performed85.71 13.62 km per week and a four-week transition period after the second competitive period. The rst preparatory period began in October through December and the second one during March through May. Competitive periods were coincident with January and February when the athletes performed cross country competitions (10,000–12,000 m approximately), and the second competitive period was in June-July when they performed track and eld competitions between 1500 and 5000 m. A GPS pack equipped with pulsometers (Polar Vantage M. Norway) was used to track the training loads during the season.

Int. J. Environ. Res. Public Health2020,17, 5833 4 of 12Int. J. Environ. Res. Public Health 2020, 17, x 4 of 12 Figure 1. Periodization and testing during the season. Table 1 summarizes training characteristics in the athletes. In addition, they performed two weekly sessions of resistance training during the whole athletics season. In general, the volume of the training was high (3 sets of 8–12 repetitions of whole-body exercises) while the intensity was low- moderate (30–70% of 1RM) depending on the period of the season. Table 1. Training characteristics in the athletes during the season. Training Load Initial 3 Months 6 Months 9 Months Total (km/week) 85.71 ± 13.62 105.9 ± 16.85 93.33 ± 14.34 74.76 ± 14.09 >VT2 (km/week) 4.29 ± 0.68 12.71 ± 2.02 18.67 ± 2.86 16.45 ± 3.10 <VT 2 (km/week) 81.43 ± 12.94 93.24 ± 14.83 74.67 ± 11.47 58.31 ± 10.99 VT2: anaerobic threshold; >VT2: intensity above anaerobic threshold; <VT2: intensity below anaerobic threshold. 2.6. Sample Collection Always at nine o’clock in the morning, to limit the impact of circadian rhythms on hormonal concentrations, after weighing the participants, ten milliliters of antecubital venous blood was drawn from each participant. Venous blood samples were obtained using EDTA as anticoagulant. Blood was immediately centrifuged at 3000 rpm during 10 min (P-selecta, MEDITRONIC) using a plastic syringe with a stainless-steel needle. The blood sample was collected in a polypropylene tube. Then, the blood sample was centrifuged at 3000 rpm for 15 min at room temperature (23 ± 1 °C) to separate plasma from erythrocytes. Plasma was placed in sterile tubes and stored at −80°C until use. 2.7. Analytical Determination. The hormone determination was carried out using the ELISA (enzyme-linked immunosorbent assay) with an ER-500 (Sinnowa, Germany), using the commercial tests for I, C, T and LH. All hormonal measurements were performed by the same technician and were made with duplicate determination. Between and within coefficients of variation for all assays were less than 10% for all biochemical analyses. 2.8. Statistical Analysis The statistical analysis was carried out with IBM SPSS Statistic software version 21.0 (IBM Co., Armonk,

the commercial tests for I, C, T and LH. All hormonal measurements were performed by the same technician and were made with duplicate determination. Between and within coefficients of variation for all assays were less than 10% for all biochemical analyses. 2.8. Statistical Analysis The statistical analysis was carried out with IBM SPSS Statistic software version 21.0 (IBM Co., Armonk, NY, USA). The results are expressed as x ± s, where x is the mean values and s is the standard deviation. All variables used in the study were checked for normality of distribution before the analyses (Kolmogorov–Smirnov tests). The data were analyzed by repeated measurements analysis Figure 1.Periodization and testing during the season. Table weekly sessions of resistance training during the whole athletics season. In general, the volume of the training was high (3 sets of 8–12 repetitions of whole-body exercises) while the intensity was low-moderate (30–70% of 1RM) depending on the period of the season. Table 1.Training characteristics in the athletes during the season. Training Load Initial 3 Months 6 Months 9 Months Total (km/week) 85.71 13.62 105.9 16.85 93.33 14.34 74.76 14.09 >VT 2(km/week) 4.29 0.68 12.71 2.02 18.67 2.86 16.45 3.10 <VT 2(km/week) 81.43 12.94 93.24 14.83 74.67 11.47 58.31 10.99 VT2: anaerobic threshold;>VT2: intensity above anaerobic threshold;<VT2: intensity below anaerobic threshold. 2.6. Sample Collection Always at nine o'clock in the morning, to limit the impact of circadian rhythms on hormonal concentrations, after weighing the participants, ten milliliters of antecubital venous blood was drawn from each participant. Venous blood samples were obtained using EDTA as anticoagulant. Blood was immediately centrifuged at 3000 rpm during 10 min (P-selecta, MEDITRONIC) using a plastic syringe with a stainless-steel needle. The blood sample was collected in a polypropylene tube. Then, the blood sample was centrifuged at 3000 rpm for 15 min at room temperature (23 1 C) to separate plasma from erythrocytes. Plasma was placed in sterile tubes and stored at 80 C until use. 2.7. Analytical Determination The hormone determination was carried out using the ELISA (enzyme-linked immunosorbent assay) with an ER-500 (Sinnowa, Germany), using the commercial tests for

the blood sample was centrifuged at 3000 rpm for 15 min at room temperature (23 1 C) to separate plasma from erythrocytes. Plasma was placed in sterile tubes and stored at 80 C until use. 2.7. Analytical Determination The hormone determination was carried out using the ELISA (enzyme-linked immunosorbent assay) with an ER-500 (Sinnowa, Germany), using the commercial tests for I, C, T and LH. All hormonal measurements were performed by the same technician and were made with duplicate determination. Between and within coe cients of variation for all assays were less than 10% for all biochemical analyses. 2.8. Statistical Analysis The statistical analysis was carried out with IBM SPSS Statistic software version 21.0 (IBM Co., Armonk, NY, USA). The results are expressed as x s, where x is the mean values and s is the standard deviation. All variables used in the study were checked for normality of distribution before the analyses (Kolmogorov–Smirnov tests). The data were analyzed by repeated measurements analysis of variance (ANOVA) with the Bonferroni post hoc test for moment/period as the categorical variable.

Int. J. Environ. Res. Public Health2020,17, 5833 5 of 12 Partial eta squared ( 2 p) was used as an e ect size measure of ANOVA. Threshold values for assessing magnitudes of standardized e ects were 2 p 0.01, 2 p 0.06 and 2 p 0.14 for small, medium and large, respectively [31]. The equality of variances between the di erences was assessed with Mauchly's test of sphericity. When sphericity was violated, Greenhouse–Geisser correctedp-values were used. Simple linear regression analysis was conducted to examine the associations between hormones and km trained per week. Ap 0.05 was considered statistically signi cant. 3. Results Table In our study, VO2max, VT2, RER, heart rate maximum (HRM), fat mass, fat-free mass and P 6 skinfolds did not show signi cant changes during the season. Weight su ered signi cant decreases (p<0.05) at 6 and 9 months compared with initial values. Table 2.Ergospirometric and body composition parameters in the athletes during the season. Parameters Initial 3 Months 6 Months 9 Months p 2 VO 2max (mL/min/kg) 68.12 4.63 67.53 9.54 68.55 6.97 68.60 7.36 0.05 VT 2(% VO 2max.) 91.02 2.43 92.43 3.59 91.02 3.08 90.96 2.07 0.06 RER 1.05 0.03 1.05 0.04 1.05 0.05 1.04 0.04 0.01 HR maximum 190.4 9.48 193.1 7.80 193.5 9.06 193.8 7.19 0.04 Weight (kg) 65.50 7.30 65.45 7.36 64.67 7.03 * 64.80 7.34 * 0.07 Fat mass (%) 8.18 1.04 8.23 1.04 8.19 1.29 8.21 1.07 0.04 Fat-free mass (kg) 60.15 6.70 60.07 6.75 59.38 6.45 59.48 6.73 0.02 P 6 skinfold (mm) 45.65 10.88 46.49 10.69 44.92 8.16 45.37 9.11 0.03 VO2max: maximum oxygen uptake; VT2: ventilatory anaerobic threshold; HR maximum: heart rate maximum; P 6: sum of 6 skinfolds *p<0.05 initial vs. 3/6/9 months; 2 p: partial eta squared. Nutritional intake of the athletes during the season is shown in Table. The athletes followed a diet using established energy and macronutrient guidelines for adequate athletic performance [32]. Energy availability (EA) is de ned as the amount of energy intake (kcal day 1 ) exercise energy expenditure (kcal day 1 )]/FFM. An appropriate energy balance equates to

partial eta squared. Nutritional intake of the athletes during the season is shown in Table. The athletes followed a diet using established energy and macronutrient guidelines for adequate athletic performance [32]. Energy availability (EA) is de ned as the amount of energy intake (kcal day 1 ) exercise energy expenditure (kcal day 1 )]/FFM. An appropriate energy balance equates to 45 calories per day per kg of FFM (kcal/kg/FFM/d) [33]. Table 3.Nutritional intake. Parameters Initial 3 Months 6 Months 9 Months Energy (kcal/d) 2855.21 511.32 2515.48 427.18 2902.37 522.62 3108.78 770.12 EA (kcal/kg/FFM/d) 43.58 4.32 41.87 3.15 48.88 5.63 52.26 4.87 HC (g/kg/d) 5.26 1.21 5.18 1.14 6.25 1.38 6.13 1.50 Proteins (g/kg/d) 1.73 0.79 1.69 0.35 1.85 0.53 1.89 0.63 Lipids (g/kg/d) 1.78 0.40 1.63 0.28 1.58 0.52 1.72 0.74 HC: carbohydrates; FFM: fat-free mass; EA: energy availability. Plasmatic concentrations of hormones are shown in Table. Table 4.Hormonal changes during the season. Parameters Initial 3 Months 6 Months 9 Months p 2 Insulin ( IU/mL) 10.25 7.99 7.81 6.15 ** 7.62 5.50 ** 9.89 5.32 * 0.51 LH (mIU/mL) 8.85 4.10 6.30 2.86 **7.59 2.32 # 7.95 3.49 # 0.29 Testosterone (ng/mL) 6.59 0.92 5.83 1.10 **6.72 0.94 # 7.01 1.50 # 0.32 Cortisol (ng/mL) 89.26 21.85 91.41 27.32 98.06 31.28 103.9 38.04 0.05 T/C 0.07 0.01 0.06 0.02 0.07 0.02 0.07 0.02 0.03 LH: luteinizing hormone; T: testosterone; C: cortisol; *p<0.05 di erences between initial vs. 3/6/9 months;**p<0.01 di erences between initial vs. 3/6/9 months; #p<0.05 di erences between 3 months vs. 6/9 months; 2 p: partial eta squared.

This study examines hormonal changes in high-level male endurance runners throughout a sports season.