Energy availability calculation
EA was calculated as EA = (EI-EEE)/FFM. Detailed instructions were given to all participants for weighing foods, using measuring tools and providing photographic evidence of everything ingested. Data was analyzed with Foodworks 9 Professional Edition (version 9.0.3973, Xyrix Software, Australia). EEE was measured by using wearable heart rate monitors during all exercise sessions (Polar V800, Polar Electro, Kempele, Finland).
Training load in stage 1 (EEE 1) was calculated from EA 1, which was 25% higher as EA 0 for each individual. Subjects exercised every day for a specific time period adjusted for their EA reduction needs. They ran or cycled at HR between 70 and 80% of their maximal HR.
Resting energy expenditure assessment
REE was measured with indirect calorimetry (V2 mask (Hans Rudolph, USA), K5 (Cosmed, Albano Laziale, Rome, Italy) with Quark 8.1. PC software support) based on the Weir equation (Weir 1949; Torstveit et al.
2019), for 30 min in a thermoneutral environment, in silence, between 6.00 and 9.00 a.m, just after the body composition measurement. Participants were in fasted state, had to refrain from physical exertion in the last 12 h and any caffeine ingestion. The final 20 min were used for REE analysis (Compher et al.
2006). For predicted REE (pREE) calculation, the Harris-Benedict equation was used (Harris and Benedict
1918). The mREE/pREE ratio was then calculated to assess energy conservation.
Blood samples
All blood samples were drawn in the morning at 9am in a fasted state to assess complete blood count, ferritin, serum iron (Fe), triiodothyronine (T3), thyroid stimulating hormone (TSH), morning testosterone, fasting insulin, insulin like growth factor 1 (IGF-1) and 9am cortisol. Blood was collected using standard clinical procedures. Haemoglobin was analysed with Sysmex XN-550 (photometric detection, EDTA tubes), iron with Cobas c501 (colorimetric analysis, serum tubes), ZSH, T3, testosterone, cortisol and ferritin with Cobas e411 (electrochemiluminescence immunoassay, serum tubes). Serum insulin level was analyzed with a double antibody RIA (serum tubes) and for IGF-1 the RIA kit (serum tubes) was used.
Psychological assessment
Mental status was assessed with the TFEQ-R18 and Well-being questionnaire (Viner et al.
2015; Jurov et al.
2020b). TFEQ-R18 and its subscale, cognitive restriction, were used to look for changes in eating behaviours as used in similar research setting before. General well-being was assessed by a simple questionnaire as recommended by Hooper and Mackinnon (Hooper and Mackinnon
1995) including six subjective ratings (fatigue, sleep, stress, muscle soreness, mood and morning erections) on a 1–5 scale. The last item about morning erection was to the original set as proposed by study on professional rugby players (McLean et al.
2010) (supplementary file 1).
To test performance, three different tests were chosen to assess vertical jump height (explosive power of lower extremities), motor task execution time (agility) and maximal aerobic capacity (aerobic endurance). Performance tests were performed in exact order for each participant: general warm-up (2 min of cycling on a stationary bike at a 50 revolutions per minute at approximately 1.5 W/kg) was followed by performing mobility exercises (athletes performed arm, hip, knee and ankle mobility exercises (10 reps each); dynamic stretches of hip flexors, knee extensors, knee flexors and ankle extensors (10 reps each); and heel raise, squat, crunch resistance exercises (10 reps each).
First, countermovement jump (CMJ) test was performed using a bilateral force plate system (Type 9260AA, Kistler Instrumente AG, Winterthur, Switzerland) with Kistler MARS software (S2P Ltd., Ljubljana, Slovenia) to acquire ground reaction force. Each subject performed three to five maximal counter movement jumps before the testing. For CMJ data were sampled at 1000 Hz, filtered using a moving average filter with 50-ms window and analyzed using the built-in module for CMJ. Test execution was supervised from the experienced researcher to improve proficiency in jumping technique [24]. Before each jump, participants were instructed to stand up straight and still on the center of the force plate with their hands akimbo. This hand position remained the same during the entire movement. From this position, participants initiated a fast downward movement until a crouching position with a knee angle of about 90°, followed by a jump for maximal height as quickly and explosively as possible. Three valid trials were performed with one-minute recovery period. The main outcome measure was CMJ height in centimeters that was calculated from the maximum velocity [25].
To asses motor task execution time, validated modified agility t-test was used, as described by Haj-Sassi, et al. (2011). The T-test was performed after 5 min rest from vertical jump testing without additional warm up. Each participant had three trials (each trail consisting of two sprints separated by 30 s rest). The rest between trials was 90 s. Cones were placed in a T-shape layout, 5 and 2.5 m apart, respectively. Subjects were instructed to sprint and change direction as fast as possible. They began with both feet 0.3 m behind the starting line (A). At their own discretion subject sprinted forward to cone B and touched the base of it with the right hand. Facing forward and without crossing feet, they shuffled to the left to cone C and touched its base with the left hand. Subjects then shuffled to the right to cone D and touched its base with the right hand. They shuffled back to the left to cone B and touched its base. Finally, subjects ran backward as quickly as possible to cross the finish line (cone A) ending the first sprint. The second sprint started after 30 s rest. Three such trials separated by 90 s rest were performed, all with verbal encouragement. The subject who crossed one foot in to each direction (firstly starting with shuffling to the left) front of the other, failed to touch the base of the cone or failed to face forward throughout, had to repeat the test. The time to complete each repetition was measured using one pair of the electronic timing system sensors (Witty Timing System, Microgate, Bolzano) mounted on tripods. They were set approximately 0.75 m above the floor positioned 2 m apart facing each other on either side of the starting line (A). The time of best repetition (seconds) and three repetition average were used in further analysis.
After 1 h of rest, endurance was measured with the incremental test to exhaustion. Heart rate, ventilatory, and gas data were collected during the incremental test with metabolic cart (K5, Cosmed, Italy). All measurements were performed in the physiological laboratory with ambient temperature of 21 °C. For measuring VO2max, the following procedure was performed. After a 15-min warm-up on bicycle set up on a cycle ergometer (Cyclus 2, Leipzig, Germany), workload constantly increased until volitional exhaustion (100 W + 20 W every minute). Lactate was analyzed in capillary blood drop from the earlobe. Samples were obtained at rest before any physical activities, at the end of the test and 5 min after the test. Lactate was analyzed with the blood lactate analyzer Biosen C_Line (EKF Diagnostics, Germany).