Procedures
Energy availability calculation
All procedures were carried during 9 days period (Fig. 1). Participant EA was measured bycompleting dietary diaries for 7 consecutive days (9). All participants received detailed information on how to complete the diary and how to weight food or measure its quantity with the help of cups and other measuring tools. They were asked to provide photographic evidence of all food and liquid ingested in that time. EI data was analyzed with Foodworks 9 Professional Edition (version 9.0.3973, Xyrix Software, Australia). During this same period EEE was estimated from heart rate using wearable heart rate monitors during all exercise sessions (Polar V800, Polar Electro, Kempele, Finland). EA was calculated as EA=(EI-EEE)/FFM (FFM = fat free mass).
Performance testing
To test performance, three different tests were chosen to assess vertical jump height (explosive power of lower extremity), motor task execution time (agility) and maximal aerobic capacity (aerobic endurance). The details of warm-up protocol, countermovement jump (CMJ), agility t-test and the incremental test can be found in the Additional file 1.
First, 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 has performed three to five maximal counter movement jumps before the testing.
Second, to asses motor task execution time, validated modified agility t-test was used, as described by Haj-Sassi, et al. (2011). The time of best repetition (seconds) were used in further analysis.
After 1 hour 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 (V2 mask (Hans Rudolph, USA), K5 (Cosmed, Albano Laziale, Rome, Italy) with Quark 8.1. PC software support) on a cycle ergometer (Cyclus 2, Leipzig, Germany).
Blood samples
On day 8, venous 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.
Body composition assessment
Body composition was assessed using tetra polar eight point tactile bioelectrical impedance device InBody 720 (Biospace, Seul, South Korea) on day 9. Prior to body composition measurement, participants received instructions how to be adequately hydrated to enable precise measurement of FFM and body fat percentage that were used in further analysis.
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 Eq. (12,13). The measurement was performed in a thermoneutral environment, in silence, between 6.00 and 9.00 a.m., after 12 hours of fasting (14). It lasted 30 min and the final 20 min were used for REE measurement (15). During measurement respiratory quotient was monitored since measures under 0.70 or above 1 suggest protocol violations or inaccurate gas measurement (15). To obtain predicted REE (pREE), a Harris-Benedict equation was used (16). The mREE/pREE ratio was then calculated for further analysis.
Psychological assessment
The Three Factor Eating Questionnaire (TFEQ-R18) and Well-being questionnaire were used for psychological assessment (17, 18). TFEQ-R18 was used to detect early changes in eating behaviors and has three subscales including cognitive restriction (CR), disinhibition and susceptibility to hunger, with higher scores indicating greater eating disturbances in participant. The subscale of interest was CR. General well-being was assessed by a simple questionnaire as recommended by 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 (20) (Additional file 2).