Dietary Intake, Serum Hormone Concentrations, Amenorrhea and Bone Mineral Density of Physique Athletes and Active Gym Enthusiasts

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Dietary Intake, Serum Hormone Concentrations, Amenorrhea and Bone Mineral Density of Physique Athletes and Active Gym Enthusiasts © 2023 by the authors. Licensee MDPI, Basel, Switzerland. Published version Mursu, Jaakko; Ristimäki, Maija; Malinen, Inga; Petäjä, Pirita; Isola, Ville; Ahtiainen, Juha P.; Hulmi, Juha J. Mursu, J., Ristimäki, M., Malinen, I., Petäjä, P., Isola, V., Ahtiainen, J. P., & Hulmi, J. J. (2023). Dietary Intake, Serum Hormone Concentrations, Amenorrhea and Bone Mineral Density of Physique Athletes and Active Gym Enthusiasts. Nutrients, 15(2), Article 382. https://doi.org/10.3390/nu15020382 2023 Citation: Mursu, J.; Ristimäki, M.; Malinen, I.; Petäjä, P.; Isola, V.; Ahtiainen, J.P.; Hulmi, J.J. Dietary Intake, Serum Hormone Concentrations, Amenorrhea and Bone Mineral Density of Physique Athletes and Active Gym Enthusiasts. Nutrients 2023,15, 382. https:// doi.org/10.3390/nu15020382 Academic Editor: Stephen Ives Received: 15 December 2022 Revised: 7 January 2023 Accepted: 8 January 2023 Published: 12 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). nutrients Article Dietary Intake, Serum Hormone Concentrations, Amenorrhea and Bone Mineral Density of Physique Athletes and Active Gym Enthusiasts Jaakko Mursu 1,*, Maija Ristimäki 2, Inga Malinen 1, Pirita Petäjä 3, Ville Isola 1, Juha P. Ahtiainen 1 and Juha J. Hulmi 1,* 1Faculty of Sport and Health Sciences, NeuroMuscular Research Center, University of Jyväskylä, 40014 Jyväskylä, Finland 2Institute of Public Health and Clinical Nutrition, University of Eastern Finland, 70211 Kuopio, Finland 3Department of Food and Nutrition, University of Helsinki, 00100 Helsinki, Finland *Correspondence: [email protected] (J.M.); [email protected] (J.J.H.) Abstract: As the diet, hormones, amenorrhea, and bone mineral density (BMD) of physique athletes (PA) and gym enthusiasts (GE) are little-explored, we studied those in 69 females (50 PA, 19 GE) and 20 males (11 PA, 9 GE). Energy availability (EA, kcal · kgFFM −1· d −1 in DXA) in female and male PA was ~41.3 and ~37.2, and in GE ~39.4 and ~35.3, respectively. Low EA (LEA) was found in 10% and 26% of female PA and GE, respectively, and in 11% of male GE. In PA, daily protein intake (g/kg body mass) was ~2.9–3.0, whereas carbohydrate and fat intakes were ~3.6–4.3 and ~0.8–1.0, respectively. PA had higher protein and carbohydrate and lower fat intakes than GE (p< 0.05). Estradiol, testosterone, IGF-1, insulin, leptin, TSH, T4, T3, cortisol, or BMD did not differ between PA and GE. Serum IGF-1 and leptin were explained 6% and 7%, respectively, by EA. In non-users of hormonal contraceptives, amenorrhea was found only in PA (27%) and was associated with lower fat percentage, but not EA, BMD, or hormones. In conclusion, off-season dietary intakes, hormone levels, and BMD meet the recommendations in most of the PA and GE. Maintaining too-low body fat during the off-season may predispose to menstrual disturbances. Keywords: fitness; low energy availability; relative energy deficiency in sport (RED-S); menstrual status; sport 1. Introduction Physique sports include various divisions from bikini athletes to bodybuilders, in all of which competitors strive to achieve an aesthetic appearance with symmetry, balance and muscle “definition” achieved with minimal fat mass. Competitive physique athletes have an off-season and a competition preparation phase. During a typical 3–8-month contest, preparation athletes aim for low body fat with negative energy balance while maintaining muscle size with resistance training and high protein intake [ 1 ]. In competitions, physique athletes are judged by their aesthetic appearance, and a relatively high amount of muscle mass and low levels of body fat are preferred [1]. Physique athletes spend most of their time in the off-season (also called ‘improvement season’), where the main goal is to increase muscle size while minimizing body fat accumulation [ 2 ]. This is achieved with resistance training providing mechanical stimuli [ 3 ] and positive energy balance, adequate protein and carbohydrate intake which support intensive exercise, muscle growth and recovery [ 4 – 6 ]. For experienced trainees, a 5–10% energy surplus, i.e., 200–300 kcal above daily maintenance, is recommended which is estimated to increase body size by 0.25% per week [ 2 ] and to minimize the adverse health effects of dieting, e.g., on hormonal functions. Not all, however, follow the nutrition recommendation in the off-season, and these strategies are common without scientific evidence, which can Nutrients 2023,15, 382. https://doi.org/10.3390/nu15020382 https://www.mdpi.com/journal/nutrients Nutrients 2023,15, 382 2 of 17 expose physique athletes to unwanted health or performance-related consequences [ 7 ]. During the off-season, obtaining optimal energy intake may be psychologically challenging, especially after a long competition preparation. In female physique athletes, body dissatisfaction and fear of gaining fat may be more common compared to men [8]. Low energy intake may negatively affect gains in muscle size, although strength gains seem to be less affected [ 9 ]. Low energy intake may also affect the health of athletes. The adequacy of energy intake can be assessed by calculating energy availability (EA) which estimates the amount of energy available, e.g., for body functions and training adaptation. EA is derived by subtracting exercise energy expenditure from energy intake and dividing by fat-free mass (FFM). EA ≥ 40–45 kcal/kg of FFM is considered to be optimal during the improvement season, supporting performance, training adaptation and health [ 10 ]. In contrary, low EA (<30 kcal/kg of FFM) is necessary for weight loss, but may negatively affect the performance, the production of various hormones and bone health [ 10 , 11 ] and thus may lead to a multisyndrome condition called relative energy deficiency in sport (RED-S) [ 10 ]. Studies investigating weight loss in these athletes have shown that low EA may suppress leptin, triiodothyronine (T3), testosterone, and estradiol concentrations, and increase the incidence of menstrual irregularities, including amenorrhea [ 12 ] markers of immunosuppression [ 13 ], and adaptive thermogenesis [ 14 ]. Furthermore, amenorrhea, which is considered to be a long-term marker for low EA [ 10 ], impairs bone health, increases the risk for bone stress injury and cardiovascular disease [ 11 ], and may thus, through these effects, alter athletes’ performance and health. These changes are reversible if the energy intake is increased to optimal levels [ 10 ]. However, the studies on these athletes have focused on competition preparation diets and not the off-season phase. Thus, there is not much evidence about the dietary habits and physiology of the athletes during this phase of their training season and how they compare to active gym enthusiasts who often have similar goals, except they may not have the aim to compete. Currently, very little is known about the dietary habits and related health of the physique athletes, especially in the off-season, and how they compare to those who actively train in the gym but do not compete in any physique or strength sports. Therefore, the aim of our cross-sectional study was to assess dietary intake, serum hormone concentrations, amenorrhea, and bone mineral density of Finnish physique athletes in the off-season and in gym enthusiasts who have similar training goals but have no experience in competing or competition preparation in physique sports. Additionally, our aim was to assess whether amenorrhea is associated with EA, body composition, training background, serum hormone concentrations, and bone mineral density. 2. Materials and Methods 2.1. Overall Approach to the Problem The current study is a sub-study of a larger Physique Athlete Study conducted by the University of Jyväskylä including a cohort collected in 2015–2016 [ 12 – 15 ] and 2019–2020 (Isola et al. submitted for revision, ClinicalTrials.gov ID: NCT04392752). The current study looked at the prevalence of energy availability and the distribution of intake of different energy nutrients. Energy availability was compared with measurements of hormone levels and bone density taken at the same time point and menstrual status in a crosssectional setting. 2.2. Participants A total of 184 healthy, physically active young females in study 1 and 49 males and 40 females in study 2, recruited by web page and social media advertisements, volunteered to participate in the study via the university web page, the governing sports body web page for physique sport, and associated social media. They were physique athletes or active gym enthusiasts volunteered to participant in either a weight loss and weight gain regimen or weight maintenance. Nutrients 2023,15, 382 3 of 17 The participants who were diagnosed with chronic diseases; reported using prescribed medications excluding birth control pills or any substances or methods prohibited by the World Antidoping Agency (WADA) such as performance-enhancing drugs; competing in junior (below 19 years of age) or master (over 40 years of age) categories; or competing at a non-drug-tested competition were excluded from the study. Participants who had competed within six months before the measurements were also excluded from the study. An online pre-study questionnaire was sent to the volunteers who claimed to meet the study’s inclusion criteria. Additional inclusion criteria for the physique athlete group were competition background in physique sports before the start of the study or registration for competitions in the following months under the International Fitness and Bodybuilding Federation (IFBB). Moreover, the inclusion criteria for the included divisions of IFBB athletes were the following: classic bodybuilding and men’s physique in males and fitness, body fitness, bikini fitness, wellness fitness and women’s physique in females. The gym enthusiasts group consisted of participants who also volunteered for the physique sport study and were actively and purposefully training to improve their physique but had not previously participated in competitions or did not intend to participate competitions in the near future (see more details of physique athletes and gym enthusiasts in Table 1). All the gym enthusiasts had a background of at least two resistance training years and they currently all train in a goal-oriented manner (improving their physique) about three to four times in the gym per week. The participants selected for the study completed an additional questionnaire that was subsequently reviewed by the study physician. The final study involved, in total, 89 participants; 61 were physique athletes and 28 gym enthusiasts. Out of 69 female participants, 50 were physique athletes and 19 were gym enthusiasts, and out of 20 male participants, 11 were physique athletes and 9 were gym enthusiasts (Table 1). Table 1. Basic information of the study participants given as mean and standard deviation. Females Males p-Values 1 PA (n= 50) GE (n= 19) PA (n= 11) GE (n= 9) Sex Athletes Sex ×Athlete Age (years) 27.7 ±4.1 26.4 ±4.2 28.0 ±5.6 32.0 ±4.7 0.085 0.260 0.022 Height (cm) 165.7 ±5.4 164.7 ±4.2 180.4 ±3.9 180.8 ±2.0 0.001 0.831 0.589 Weight (kg) 65.0 ±6.9 63.5 ±5.5 89.1 ±8.8 86.1 ±6.0 0.001 0.196 0.685 BMI (kg/m2) 23.6 ±1.7 23.4 ±1.7 27.4 ±2.7 26.3 ±1.7 0.001 0.172 0.429 BF (%) 23.4 ±5.5 23.5 ±5.8 14.9 ±4.2 15.7 ±6.8 0.001 0.814 0.798 FFM (kg) 50.4 ±4.7 49.3 ±4.2 76.7 ±9.1 72.4 ±3.8 0.001 0.054 0.255 RT years 4.1 ±1.9 3.6 ±1.2 6.5 ±2.3 6.4 ±3.7 0.151 0.420 0.546 Training frequency 7.6 ±3.0 8.0 ±4.2 5.5 ±1.6 4.6 ±1.8 0.688 0.814 0.288 Total training volume (h/week) 6.8 ±2.5 5.1 ±2.4 5.0 ±1.3 4.6 ±2.2 0.262 0.099 0.688 RT frequency 4.6 ±0.9 3.5 ±1.8 4.7 ±0.8 4.1 ±1.1 0.194 0.007 0.472 MET (h/week) 41.2 ±16.9 46.1 ±35.3 26.6 ±8.5 29.5 ±21.7 0.910 0.363 0.710 EEE (kcal) 386 ±175 418 ±317 340 ±114 361 ±261 0.204 0.506 0.796 EI (kJ) 10,230 ± 1820 9846 ±2102 13,250 ± 1494 12,170 ±942 0.001 0.124 0.459 EI (kcal) 2444 ±435 2352 ±502 3165 ±357 2907 ±225 0.001 0.124 0.460 Protein (E%) 32.6 ±6.8 28.1 ±5.3 32.1 ±6.3 29.6 ±5.0 0.463 0.041 0.591 Nutrients 2023,15, 382 4 of 17 Table 1. Cont. Females Males p-Values 1 PA (n= 50) GE (n= 19) PA (n= 11) GE (n= 9) Sex Athletes Sex x Athlete Protein (g/day) 195.7 ±40.8 162.9 ±33.0 251.1 ±35.8 214.8 ±38.5 0.001 0.001 0.844 CHO (E%) 38.0 ±7.2 34.2 ±5.5 47.7 ±9.5 40.6 ±8.1 0.011 0.004 0.422 CHO (g/day) 233.2 ±67.6 201.2 ±50.8 376.1 ±73.3 295.1 ±61.7 0.001 0.001 0.165 Fat (E%) 25.0 ±7.1 33.0 ±6.4 21.1 ±3.7 26.7 ±6.0 0.032 0.001 0.466 Fat (g/day) 67.0 ±19.9 86.0 ±24.3 73.1 ±5.8 86.2 ±19.5 0.516 0.003 0.566 PA = physique athletes; GE = gym enthusiasts; BMI = body mass index; BF = body fat; FFM = fat-free mass, RT = resistance training, E = energy, CHO = carbohydrates. Training refers to all goal-oriented exercise such as resistance training or aerobic training. Training frequency refers to the number of training sessions/week. 1 pfor difference using general linear model. All participants gave their written informed consent for inclusion before they participated in the study (first cohort) and the Ethics Committee of the Central Finland Health Care District (19U/2018), Finland (the second cohort). The latter cohort was also registered at ClinicalTrials.gov ID: NCT04392752. The studies were conducted according to the guidelines of the Declaration of Helsinki. The participants were given comprehensive explanations regarding the study design, protocols, and possible risks. All participants gave written informed consent. The participants were given identification numbers, and the research group was blinded throughout the study. 2.3. Study Design The study was a cross-sectional analysis of an observational study. We provided no intervention, and all groups followed their own preferred diet and exercise regimen. The time point for the measurement was during the off-season before a possible competition season dieting phase. The participants arrived at the laboratory between 07.00 and 09.00 am after at least eight hours of fasting and after instruction to sleep at least eight hours and abstain from alcohol and caffeine for 12 h, and exercise for 24 h. If participants traveled over 50 km to the laboratory, they were provided a hotel room for the night before the measurement day. Participants were advised to avoid physical activity, such as walking, jogging, and cycling, on the morning of assessment. The participants from the hotel were transported to the laboratory by car. In comparison, participants who lived closer than 50 km to our laboratory were advised to come via car or public transportation to the tests. 2.4. Estimation of Energy Availability EA was calculated as (energy intake—exercise energy expenditure)/FFM. In the study, body composition was measured by DXA, InBody720 bioimpedance and skinfold measurement, of which DXA was chosen as the body composition method for EA (more below). Study participants kept a 4-day food diary or provided information of their current dietary program by their coaches that they strictly followed. These included dietary supplements and all beverages, except water. The intake of protein and other energy providing supplements was included in the calculations of dietary energy and nutrients. The food diaries and the completed nutrition programs were analyzed by nutrient analysis software (Aivodiet, Flow-team Oy, Oulu, Finland) that is based on the national Food Composition Database Fineli in Finland maintained by the Finnish Institute for Health and Welfare. The software uses Fineli, which is a database maintained by the Finnish Institute for Health and Welfare (THL). Exercise energy expenditure was estimated using exercise diaries and exercise programs. Subjects exercised according to their own exercise programs throughout the study period. They were asked to report all exercise in the exercise diary provided to them. The Nutrients 2023,15, 382 5 of 17 exercise diaries were used to determine (1) the number of resistance exercise workouts per week and type of workouts (upper body/lower body/whole body) and (2) the number of aerobic workouts per week, type and duration. For some subjects, exercise data were collected directly from their exercise programs. For each exercise, a metabolic equivalent (MET) value was determined according to Ainsworth et al. [ 16 , 17 ] The MET coefficient indicates the multiple of the energy expenditure compared to the resting energy expenditure. In this study, the MET values for resistance exercise were used as follows: lower-body exercise 6, upper-body exercise 4 and total-body exercise 5 MET. Resistance exercise duration was set at 1 h for those who did not report exercise duration separately, referring to previous studies in physique athletes/bodybuilders [18]. Exercise-induced energy expenditure was calculated by multiplying the MET value by the duration of the exercise to obtain METh values for each exercise. The METh values for each exercise in a week were summed and divided by seven to give the average METh value for a single day’s exercise. This number was multiplied by the body weight of each participant to obtain the energy expenditure in kilocalories (kcal) spent due to exercise. 2.5. Body Composition and Bone Parameters Body composition was estimated by dual-energy X-ray absorptiometry (DXA, Lunar Prodigy Advance EnCore version 14.10.022, GE Medical Systems—Lunar, Madison, WI, USA) after overnight fasting. The participants were measured with their arms at their sides with minimal clothing (i.e., underwear). Their legs were secured by non-elastic straps at the ankles. All metal objects were removed from the participant before the scan. DXA measurements were used to record participants’ weight, fat percentage, fat-free mass (FFM), bone mineral density (BMD) and z-score. The z-scores compare BMD of individuals to age and sex-matched controls and was thus used in the present study as a measure of BMD. A z-score below − 2.0 is low bone density, being, thus, below the expected range for age and sex, but in athletes, even −1.0 has been considered to warrant further investigation [19]. 2.6. Serum Hormones and Menstrual Status Venous blood samples were obtained from the antecubital vein into serum tubes (Venosafe; Terumo Medical Co., Leuven, Hanau, Belgium) using standard laboratory procedures. Samples were stored in room temperature for 30 min, after being centrifuged at 3500 × rpm for 10 min (Megadure 1.0 R Heraeus; DJB Lab Care, Hanau, Germany). Free thyroxine (T4), free triiodothyronine (T3), thyroid-stimulating hormone (TSH) insulin and estradiol were analyzed from serum with the Immunolite 2000 XPi, immunoassay system (Seimen Healtineers, Erlangen Germany) using Immulite ® 2000 Free T3 (L2KF32), Immulite®2000 Free T4 (L2KFT42), and Immulite®2000 Third Generation TSH (L2KTS2), insulin Immulite ® 2000 (L2KIN-19), and estradiol Immulite ® 2000 (L2KE2-17) commercial kits. Serum leptin was analyzed with the Dynex Ds 2 ELISA processing System (DYNEX Technologies, Chantilly, VA, USA) using a commercial kit (Human Leptin ELISA, Clinical Range, REF RD191001100. These hormones are routinely analyzed in our laboratory and day-to-day reliability (CV%) for all of these hormones in our laboratory is <8%. Menstrual status and the possible use of hormonal contraception (oral hormonal contraceptives or intrauterine device) was investigated using questionnaires. The participants returned the questionnaire at the time of measurement. 2.7. Statistical Analysis The females and males were divided into three categories according to EA: (1) <30 kcal/kg FFM, (2) 30–45 kcal/kg FFM and (3) >45 kcal/kg FFM and (1) <30 kcal/kg FFM, (2) 30–40 kcal/kg FFM and (3) >40 kcal/kg FFM, respectively [ 11 ]. Female participants were divided into three categories according to menstrual status: (1) no menstrual period for more than 6 months; (2) at least one menstrual period in the last 6 months; (3) no menstrual period for more than 6 months but using hormonal Nutrients 2023,15, 382 6 of 17 contraception and (4) at least one menstrual period in the last 6 months and using hormonal contraception. The study data were analyzed using IBM SPSS statistical software 28.0 (Armonk, New York, USA) and the ones visualized also with GraphPad Prism version 9.4.1 (GraphPad Software, San Diego, California, USA), with a p-value < 0.05 as the limit of statistical significance. Descriptive analyses were used to examine baseline data, health variables, energy nutrients, energy intake and exercise data. Results were described using means and standard deviations. The effects of sex and group on age, anthropometrics, training volumes, exercise duration, MET hours, exercise energy expenditure, dietary intakes, EA, hormones and BMD were examined using two-way ANOVA. Percentages were used to describe categorical variables and chi-square tests were used in statistical comparison. Differences between groups were examined using a two-tailed t-test for normally distributed variables and a Mann–Whitney U test, the unpaired counterpart of a two-tailed t-test for non-normally distributed variables. The Kolmogorov–Smirnov and Shapiro–Wilk tests were used to test the normality of the variables. The association of energy availability with bone density and hormone levels was examined using Pearson’s correlation coefficient and variables where a correlation was found were tested using linear regression analysis. 3. Results BMI and fat-free mass (FFM) were higher and body fat percentage lower in males than in females (Table 1). Within sexes, physique athletes and gym enthusiasts were similar in age, height, weight, BMI, body fat and FFM, although higher FFM in physique athletes reached borderline significance when compared to gym enthusiasts (p= 0.054, Table 1). The number of training sessions per week, total amount of physique training per week, and total METs were higher in females, but the number of training years was shorter than in male physique athletes (p< 0.05, Table 1). Resistance training volume and total training volume tended to be higher in physique athletes compared to gym enthusiasts (Table 1), mainly due to the higher volumes in female physique athletes when compared gym enthusiasts (p< 0.05). No difference was found in the number of training sessions, training years, MET, or exercise energy expenditure between athletes and gym enthusiasts (Table 1). The EA was higher in females than in males (p= 0.020), while no difference was found between athletes and gym enthusiasts in EA (p= 0.392) (Figure 1A). The EA was in the optimal range (in females >45 kcal/kg FFM and in males >40 kcal/kg FFM) in 30% of the female and in 18% of the male physique athletes, and in 32% of the female and in 11% male gym enthusiasts, respectively. The prevalence of low energy availability (LEA) in female physique athletes was 10%, while in male physique athletes, no LEA was found (p= 0.274). In female and male gym enthusiasts, the prevalence of LEA was 26% and 11%, respectively (p= 0.360). Relative to bodyweight, physique athletes had higher dietary intakes of protein and carbohydrates, and a lower intake of fat (p< 0.05) than gym enthusiasts (Figure 1B–D). Female athletes had a lower intake of carbohydrates per body weight (p= 0.015), but higher dietary fat intake per body weight (p= 0.013) compared to male athletes, while in gym enthusiasts, carbohydrate intake did not differ between sexes. Within sexes, female physique athletes had higher intake of protein per body weight (p= 0.005) and lower intake of fat per body weight (p= 0.002) than female gym enthusiasts. The findings were similar, although not statistically significantly different, between male athletes and gym enthusiasts. Nutrients 2023,15, 382 7 of 17 Nutrients 2023, 15, x FOR PEER REVIEW 7 of 16 Figure 1. Energy availability (A) and macronutrient intakes (B–D) in physique athletes (PA) and gym enthusiasts (GE). 2 × 2 ANOVA (main and interaction effects) p-values are shown as text above the bars and possible post hoc (Tukey’s test) differences in mean values between individual groups. * = p < 0.05 and ** = p < 0.01. Ath = athlete status (PA or GE). Dashed lines for EA (A) show low (<30 kcal/kg FFM) and optimal values (> 40/45 kcal/kg FFM) and for macronutrient intakes (B–D) recommended values for bodybuilders in the off-season [2]. Due to the uncertainty whether dietary and exercise parameters are able to estimate EA, we next analyzed blood hormones [20]. No difference was found in estradiol, testosterone, IGF-1, insulin, leptin, TSH, T4, T3 or cortisol concentrations between physique athletes and gym enthusiasts within sexes (Figure 2). In athletes and gym enthusiasts, the hormone profiles were similar and in most, the concentrations were within reference values. The exception was cortisol in females, which was higher than reference range in 48% of the females. When comparing female athletes (46%) and gym enthusiasts (53%), the proportion of the females with higher cortisol concentrations than Figure 1. Energy availability ( A ) and macronutrient intakes ( B – D ) in physique athletes (PA) and gym enthusiasts (GE). 2 × 2 ANOVA (main and interaction effects) p-values are shown as text above the bars and possible post hoc (Tukey’s test) differences in mean values between individual groups. * = p< 0.05 and ** = p< 0.01. Ath = athlete status (PA or GE). Dashed lines for EA ( A ) show low (<30 kcal/kg FFM) and optimal values (>40/45 kcal/kg FFM) and for macronutrient intakes (B–D) recommended values for bodybuilders in the off-season [2]. Due to the uncertainty whether dietary and exercise parameters are able to estimate EA, we next analyzed blood hormones [ 20 ]. No difference was found in estradiol, testosterone, IGF-1, insulin, leptin, TSH, T4, T3 or cortisol concentrations between physique athletes and gym enthusiasts within sexes (Figure 2). In athletes and gym enthusiasts, the hormone profiles were similar and in most, the concentrations were within reference values. The exception was cortisol in females, which was higher than reference range in 48% of the females. When comparing female athletes (46%) and gym enthusiasts (53%), the proportion of the females with higher cortisol concentrations than reference range were similar. In Nutrients 2023,15, 382 8 of 17 other hormones, the proportions out of reference were smaller and similar between athletes and gym enthusiasts. Nutrients 2023, 15, x FOR PEER REVIEW 8 of 16 reference range were similar. In other hormones, the proportions out of reference were smaller and similar between athletes and gym enthusiasts. Figure 2. Serum hormone concentrations (A-I) in physique athletes (PA) and gym enthusiasts (GE). 2 × 2 ANOVA (main and interaction effects) p-values are shown as text above the bars and possible post hoc (Tukey’s test) differences in mean values between individual groups. * = p < 0.05 and ** = p < 0.01. Dashed lines indicate national reference ranges except for leptin, of which reference values are BMI- and sex-dependent and thus not shown. The data were complete (n = 89), except for TSH (n = 88), insulin (n = 85), testosterone (n = 87) and estradiol (n = 88). Long-term LEA can lead to low bone mineral density (BMD) [20]. Bone mineral density was similar in female and male physique athletes when compared to gym enthusiasts (Figure 3A), while in males, the BMD (1.33 ± 0.08 g/cm 2 ) was higher than in Figure 2. Serum hormone concentrations ( A – I ) in physique athletes (PA) and gym enthusiasts (GE). 2 × 2 ANOVA (main and interaction effects) p-values are shown as text above the bars and possible post hoc (Tukey’s test) differences in mean values between individual groups. * = p< 0.05 and ** = p< 0.01. Dashed lines indicate national reference ranges except for leptin, of which reference values are BMI- and sex-dependent and thus not shown. The data were complete (n= 89), except for TSH (n= 88), insulin (n= 85), testosterone (n= 87) and estradiol (n= 88). Nutrients 2023,15, 382 15 of 17 11. Melin, A.K.; Heikura, I.A.; Tenforde, A.; Mountjoy, M. Energy Availability in Athletics: Health, Performance, and Physique. Int. J. Sport Nutr. Exerc. Metab. 2019,29, 152–164. [CrossRef] [PubMed] 12. Hulmi, J.J.; Isola, V.; Suonpää, M.; Järvinen, N.J.; Kokkonen, M.; Wennerström, A.; Nyman, K.; Perola, M.; Ahtiainen, J.P.; Häkkinen, K. 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