Effects of Calorie Restriction and Cold Stimuli on Health-related Indicators, Cognitive and Motor Functions

Study Overview
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Brief Summary
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The goal of this clinical trial was to identify if duration of acute calorie restriction (CR)/fasting or combination CR with cold stimuli have any effects on mental and physical health-related markers, and to clarify if different fasting strategies have any effect on cognitive and motor functioning efficiency in different genders. The main questions it aims to answer were: Does fasting duration have any effect on fasting evoked responses? Does cold interventions can modulate fasting evoked responses? Does sex have any effect on acute fasting evoked responses? For the first part, participants were randomly classified into 4 groups: two experimental groups: 2-days of CR (0 kcal diet) and 6-days of CR (0 kcal diet), and two control groups: 2-days or 6-days usual diet. For the second part, participants were randomly assigned to undergo the following conditions: 2-days of CR with two 10-min whole-body cold-water immersions on separate days, 2-days of CR without cold-water immersion, 2-days without CR with two 10-min whole-body cold-water immersions on separate days, or 2-days of the usual diet without cold-water immersion in a randomized crossover fashion. Changes in anthropometric characteristics, perceived stress, metabolism, overall health (total blood count, sex hormones, etc.), psycho-emotional state, cognitive and motor functions were examined.

Detailed Description
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The goal of this clinical trial was to identify if duration of acute calorie restriction (CR)/fasting or combination CR with cold stimuli have any effects on mental and physical health-related markers, and to clarify if different fasting strategies have any effect on cognitive and motor functioning efficiency in different genders. At first study part, participants were randomly classified into 4 groups: two experimental groups: 2-days of calorie restriction (CR) (0 kcal diet) and 6-days of CR (0 kcal diet), and two control groups: 2-days or 6-days usual diet. In the experimental groups, the participants were instructed to follow a prescribed zero-calorie diet with water provided ad libitum over a period of 2 or 6 days. In the control groups, the participants were instructed to maintain their previous eating habits for 2 or 6 days. Body weight and composition, heart rate variability, pulmonary gas exchange, blood pressure, and body temperature were assessed. Subsequently, participants completed self-assessment questionnaires, and then saliva samples were collected for further cortisol and sex hormones analysis, also capillary and venous blood samples were taken for ketone, glucose, insulin, lipid profile, catecholamines, brain-derived neurotrophic factor, glucagon, interleukin-10, metabolites of the kynurenines pathway and complete blood count concentrations evaluation. Then, the manual dexterity, and the cognitive and neuromuscular functions were assessed. The same measurements were repeated before each trial, after each trial and after 1 week. At second part, participants were randomly assigned to undergo the following conditions: 2-days of CR with two 10-min whole-body cold-water immersions on separate days, 2-days of CR without cold-water immersion, 2-days without CR with two 10-min whole-body cold-water immersions on separate days, or 2-days of the usual diet without cold-water immersion in a randomized crossover fashion. The same measurements described in first part were repeated before each and after each condition.

Official Title
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Effects of 2-day and 6-day Calorie Restriction and Whole-body Cooling Strategies on Health-related Indicators, Cognitive and Motor Functions

Conditions
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Calorie Deficiency, Fasting, Cold Exposure

Intervention / Treatment
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* Other: 2-days of zero calorie restriction and whole-body cold-water immersion
* Other: 6-days of calorie restriction
* Other: 2-days of zero calorie restriction without whole-body cold-water immersion
* Other: 2-days of usual diet with whole-body cold-water immersion


Participation Criteria
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Eligibility Criteria
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Inclusion criteria: body mass index 19.5-29.9 kg/m2, age 18-40 years, no excessive regular moderate or vigorous physical activity, i.e., ≥3 times per week, non-smokers, no medications that could affect the results of experiment, no history of any eating, oncological, metabolic, cardiovascular, skeletal, neuromuscular, pulmonary disorders or diseases, mental disabilities or conditions that could be worsened by exposure to acute cold (14°) water or fasting and affect experimental variables, no history of alcoholism or dependence on psychotropic drugs, no blood/needle phobia, no involvement in any temperature-manipulation program or extreme temperature exposure for ⩾3 months, Exclusion criteria: smoking, were in a weight-reduction program, followed a low-carbohydrate diet.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 40 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: This project consisted of two parts. For the first part was used randomized parallel model, and we aimed to investigate fasting duration effect, whereas for the second part was used randomized crossover model and we aimed to investigate if cold exposure can modify fasting evoked effects on health-related indicators, cognition and motor function.
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: 2-days of calorie restriction with whole body cooling<br>Healthy young subjects participated in a 2-day CR (0 kcal with water provided ad libitum ) with two 10-min whole body cooling practices on separate days. All participants were asked to not perform excessive sports activities while the research was ongoing, not to be involved in any temperature-manipulation program or extreme temperature exposure for ⩾3 months; do not use any medications that could affect experimental data. | Other: 2-days of zero calorie restriction and whole-body cold-water immersion<br>* The 2-days (48 hours) of zero-calorie restricted diet (with water provided ad libitum) with two whole-body cold-water immersion sessions on separate days. During cooling procedures, the participant was immersed in 14° water bath in semi recumbent position up to the level of the manubrium for 10 min.<br>|
| Experimental: 6-days of calorie restriction<br>Healthy young subjects participated in 6-day CR (0 kcal with water provided ad libitum). All participants were asked to not perform excessive sports activities while the research was ongoing, not to be involved in any temperature-manipulation program or extreme temperature exposure for ⩾3 months; do not use any medications that could affect experimental data. | Other: 6-days of calorie restriction<br>* The 6-days (144 hours) of prescribed zero-calorie prescribed diet with water provided ad libitum.<br>|
| No Intervention: 2-days of the usual diet (control)<br>During the control trial, the healthy young subjects were instructed to maintain their previous eating habits for 2 days. All participants were asked to not perform excessive sports activities while the research was ongoing, not to be involved in any temperature-manipulation program or extreme temperature exposure for ⩾3 months, CR programmes; do not use any medications that could affect experimental data. | |
| No Intervention: 6 days of the usual diet (control)<br>During the control trial, the healthy young subjects were instructed to maintain their previous eating habits for 6 days. All participants were asked to not perform excessive sports activities while the research was ongoing, not to be involved in any temperature-manipulation program or extreme temperature exposure for ⩾3 months, CR programmes; do not use any medications that could affect experimental data. | |
| Experimental: 2-days of calorie restriction without whole-body cooling<br>Healthy young subjects participated in a 2-day CR (0 kcal with water provided ad libitum ) without whole body cooling. All participants were asked to not perform excessive sports activities while the research was ongoing, not to be involved in any temperature-manipulation program or extreme temperature exposure for ⩾3 months; do not use any medications that could affect experimental data. | Other: 2-days of zero calorie restriction without whole-body cold-water immersion<br>* 2 days (48 hours) of prescribed zero-calorie diet with water provided ad libitum .<br>|
| Active Comparator: 2-days of usual diet with whole-body cooling<br>Healthy young subjects participated two 10-min whole body cooling practices on separate days and were instructed to maintain their previous eating habits for 6 days. All participants were asked to not perform excessive sports activities while the research was ongoing, not to be involved in any temperature-manipulation program or extreme temperature exposure for ⩾3 months, CR programmes; do not use any medications that could affect experimental data. | Other: 2-days of usual diet with whole-body cold-water immersion<br>* Two whole-body cold-water immersion sessions on separate days. During cooling procedures, the participant was immersed in 14° water bath in semi recumbent position up to the level of the manubrium for 10 min. During intervention, participants were instructed to maintain their previous eating habits.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in body mass and body composition (kg) | Body mass and composition (in kg) was evaluated using Tanita Body Composition Analyzer (Japan). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in body mass index (kg/m2) | The body mass index (in kg/m2) was defined as the body mass divided by the square of the body height. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in substrate oxidation | Oxygen consumption and carbon dioxide output every 5 s on a breath-by breath basis using an Oxycon Mobile spirometry system (Oxygen Mobile, Jaeger/ VIASYS Healthcare, Germany) was measured at rest, and the respiratory quotient (RQ=VCO2/VO2) was computed to determine substrate utilisation. The RQ values for fat was assumed as 0.7, for protein was assumed as 0.8 and for carbohydrate was assumed as 1.0. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in energy expenditure (kcal/day) | Resting energy expenditure (REE) using Weir equation modified Weir equation: REE (kcal/day)=[3.941(oxygen consumption) + 1.106(carbon dioxide output)] x 1440 | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in body temperature (°C) | Rectal temperature (°C) was measured using a thermocouple (Rectal Probe, Ellab, Denmark) inserted to a depth of 12 cm past the anal sphincter, skin temperature (°C) was measured with thermistors (Skin/Surface Probe, DM852, Ellab) at three sites: back, thigh, and forearm, and right lateral gastrocnemius muscle temperature (°C) was measured using a needle microprobe (MKA; Ellab). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in heart rate variability (ms) | R-R intervals (in ms) in supine resting conditions were recorded using a Polar HR sensor (Finland) and and simultaneously transferred to Polar Pro Trainer 5 software (Finland). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in heart rate variability (time domain) (ms) | Heart rate variability data were analyzed using Kubios HR Variability Analysis software (Finland). In the time domain that reflects general heart rate variability (HRV), the standard deviation of normal-to-normal intervals (SDNN; estimate of overall HRV) and the root mean square of successive differences (RMSSD; estimate of short-term components of HRV) were assessed (in ms). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in heart rate variability (time domain) (Ln) | Heart rate variability data were analyzed using Kubios HR Variability Analysis software (Finland). In the time domain that reflects general heart rate variability (HRV), the standard deviation of normal-to-normal intervals (SDNN; estimate of overall HRV) and the root mean square of successive differences (RMSSD; estimate of short-term components of HRV) were assessed and logarithmically transformed (Ln) to correct the skewness of distribution. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in heart rate variability (frequency domain) (ms2) | Heart rate variability data were analyzed using Kubios HR Variability Analysis software (Finland). In the frequency domain that measures the more specific contribution of the autonomic nervous system branch, we used the fast Fourier transform to assess low-frequency (LF; estimates sympathetic and parasympathetic activity) and high-frequency (HF; estimates parasympathetic activity) powers in absolute units (in ms2). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in heart rate variability (frequency domain) (Ln) | Heart rate variability data were analyzed using Kubios HR Variability Analysis software (Finland). In the frequency domain that measures the more specific contribution of the autonomic nervous system branch, we used the fast Fourier transform to assess low-frequency (LF; estimates sympathetic and parasympathetic activity) and high-frequency (HF; estimates parasympathetic activity) powers were assessed and logarithmically transformed (Ln) to correct the skewness of distribution. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in heart rate variability (frequency domain) (nu) | Heart rate variability data were analyzed using Kubios HR Variability Analysis software (Finland). In the frequency domain that measures the more specific contribution of the autonomic nervous system branch, we used the fast Fourier transform to assess low-frequency (LF; estimates sympathetic and parasympathetic activity) and high-frequency (HF; estimates parasympathetic activity) powers in normalized units (in nu). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in heart rate (bpm) | Heart rate (in bpm) was recorded using a heart rate sensor with a chest strap (Polar, Finland) in laying position at rest. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in blood pressure (mmHg) | Resting systolic and diastolic blood pressure (in mmHg) was measured using a digital electronic blood pressure monitor (Microlife, Switzerland) | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in perceived stress | Perceived stress was evaluated with visual analog scales (VAS) ranging from 0 (no stress) to 100 (the highest stress imaginable).according to how participants feel right now. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in appetite sensations | Appetite sensations (hunger and fullness) were evaluated with VAS ranging from 0 (I am not hungry at all/not at all full) to 100 (I have never been more hungry/totally full) according to how participants feel right now. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in mood state | Mood state was evaluated with Brunel mood scale according to how participants feel right now. The scale consists of 24 items divided into six subscales: anger, confusion, depression, fatigue, tension, and vigor. The items are answered on a 5-point scale, and each subscale, with four relevant items, are summed to produce a raw score in the range of 0-16, where a higher score indicates greater endorsement of the specific mood state. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in venous complete blood count (10^9/L) | Venous complete blood count with 5 different white blood count (WBC) components (absolute neutrophils, lymphocytes, monocytes, eosinophils, basophils) analysis (in 10^9/L) was performed using an automated Mythic 60 hematology analyzer (Switzerland). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in venous complete blood count (%) | Venous complete blood count with 5 different white blood count (WBC) components (absolute neutrophils, lymphocytes, monocytes, eosinophils, basophils) analysis (in %) was performed using an automated Mythic 60 hematology analyzer (Switzerland). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in serum female sex hormones concentration (pg/mL) | The venous serum 17beta-estradiol and progesterone (in pg/mL) were measured using enzyme-linked immunosorbent assay kits and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in serum female sex hormones concentration (mIU/ml) | The venous serum follicle stimulating and luteinizing hormones (in mIU/ml) were measured using enzyme-linked immunosorbent assay kits and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in serum brain-derived neurotrophic factor concentration (pg/ml) | The venous serum brain-derived neurotrophic factor (in pg/ml) was measured using enzyme-linked immunosorbent assay kits (Cat.No. DBD00; R&D Systems, Emeryville, USA) and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in serum interleukin-10 concentration (pg/ml) | The venous serum interleukin (in pg/ml) was measured using enzyme-linked immunosorbent assay kits (Cat. No. 30147233; IBL International GmBH, Germany) and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in serum insulin concentration (μIU/ml) | The venous serum insulin concentrations (in μIU/ml) were measured using enzyme-linked immunosorbent assay kits (Cat. No. E-EL-H2237, Elabscience, China) and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in serum glucagon concentration (pg/ml) | The venous serum glucagon concentrations (in pg/ml) were measured using enzyme-linked immunosorbent assay kits (DIAsource ImmunoAssays S.A.,Belgium) and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in plasma catecholamines concentration (ng/ml) | The venous plasma adrenaline and noradrenaline concentrations (in ng/ml) were measured using enzyme-linked immunosorbent assay kits (Cat. No. RE59242, IBL International GmbH, Germany) and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in venous glucose concentration (mmol/l) | The venous glucose concentration (in mmol/l) was measured in venous blood samples using a Glucocard X-mini plus glucose analyser (Arkray, Japan). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in insulin sensitivity | An oral glucose insulin sensitivity [OGIS] index derived from an oral glucose tolerance test was calculated. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in plasma metabolites of the kynurenine pathway (μm) | An ultra-performance liquid chromatography-tandem mass spectrometry system (UPLC-MS/MS) to measure venous plasma levels of tryptophan, kynurenine, kynurenic acid, 3-hydroxy-kynurenine, quinolinic acid, nicotinamide and picolinic acid (in μm). The UPLC-MS/MS system used a Xevo TQ-XS triple quadrupole mass spectrometer (Waters) with a Z-spray electrospray interface, and the system was operated in electrospray positive multiple reaction monitoring mode. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in salivary cortisol concentration (µg/dl) | The saliva samples were collected to measure cortisol level (in µg/dl) using a enzyme-linked immunosorbent assay (ELISA) kits and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in salivary testosterone concentration (µg/dl) | The saliva samples were collected to measure testosterone level (in µg/dl) using a enzyme-linked immunosorbent assay (ELISA) kits and a Spark multimode microplate reader (Tecan, Austria). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in capillary lipid profile (mmol/l) | The capillary blood samples were collected from finger to measure lipid profile (in mmol/l) (total cholesterol, high density and low density cholesterol, triglycerides) using a CardioChek PA analyzer (USA). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in manual dexterity performance (sec) | The Grooved Pegboard was used to evaluate the ability to coordinate the fingers and manipulate objects promptly in time twice (in sec). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in information processing (μV) | Using a 32-channel Standard Brain Cap (EasyCap GmbH, Germany), event-related potentials (ERPs) during oddball tasks by two modalities (auditory and visual) were recorded. Peak amplitudes (μV) of the N1, N2 and P3 at three sites (Fz, Cz, and Pz) were defined. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in information processing (ms) | Using a 32-channel Standard Brain Cap (EasyCap GmbH, Germany), event-related potentials (ERPs) during oddball tasks by two modalities (auditory and visual) were recorded. Latencies (ms) of the N1, N2 and P3 at three sites (Fz, Cz, and Pz) were defined. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in brain neural network activity (µV2) | Resting electroencephalography was recorded using a 32-channel Standard Brain Cap (EasyCap GmbH, Germany). Participant's data were averaged across the epochs for Fz, Cz and Pz electrodes, and mean absolute power (in µV2) was computed for theta (4-8 Hz), alpha (8-12 Hz) and beta (12-30 Hz) frequency band. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in cognitive performance (ms) | Two oddball tasks were used in this study: in one task, visual stimuli were presented, and in the other task, auditory stimuli were presented. Reaction times (in ms) were measured. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in cognitive performance (%) | Two oddball tasks were used in this study: in one task, visual stimuli were presented, and in the other task, auditory stimuli were presented. Accuracy of response to the target stimulus (in %) were measured. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in reflexes (mV) | Soleus H-reflexes, V-waves and M-waves were evoked by 0.5 ms square-wave pulses using a high-voltage stimulator (Digitimer, UK). The amplitudes (in mV) of the electrical evoked reflexes were evaluated. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in reflexes (ms) | Soleus H-reflexes, V-waves and M-waves were evoked by 0.5 ms square-wave pulses using a high-voltage stimulator (Digitimer, UK). The latencies (in ms) of the electrical evoked reflexes were evaluated. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in muscle activity (mV) | Tibial muscles electromyographic (EMG) amplitude (in mV) parameters of muscular activity were measured using surface EMG (Biometrics, UK) thorough neuromuscular function assessment. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in muscle activity (Hz) | Tibial muscles electromyographic (EMG) frequency (in Hz) parameters of muscular activity were measured using surface EMG (Biometrics, UK) thorough neuromuscular function assessment. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in voluntary torque (Nm) | Isometric and isokinetic voluntary torques (in Nm) of the ankle plantar flexion/dorsiflexion muscles were measured using an isokinetic dynamometer (Biodex Medical Systems, USA). | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in involuntary torque (Nm) | Involuntary torque of the ankle plantar flexion muscles were measured using an isokinetic dynamometer (Biodex Medical Systems, USA) and a high-voltage stimulator (Digitimer DS7A, Digitimer, UK). Peak torques (in Nm) induced by electrical stimulation at 20 Hz,at 100 Hz, and at TT100 were measured. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in muscle contraction and relaxation (ms) | The contraction and half-relaxation time (in ms) were measured in resting TT100 contractions. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in central activation ratio (%) | To evaluate central activation ratio (CAR), a TT-100 Hz stimuli was superimposed on the maximal voluntary contraction (MVC), and the CAR was computed using the following equation: CAR = MVC/(MVC+TT-100 Hz) × 100%, where where a CAR of 100% indicates complete activation of the exercising muscle and a CAR < 100% indicates central activation failure or inhibition. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in reactive strength | The drop jump test on a force mobile platform (AccuPower, AMTI, USA) was used to evaluate reactive-strength. Jumps were performed while holding the hands of the subject on the hips were requested jump as fast as possible after the drop off from the platform and make sure that the jump is the highest possible. Reactive strength index as jump height / time to take off was calculated. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in jump performance (cm) | The drop jump test on a force mobile platform (AccuPower, AMTI, USA) was used to evaluate jump performance. Jumps were performed while holding the hands of the subject on the hips were requested jump as fast as possible after the drop off from the platform and make sure that the jump is the highest possible. Jump height (in cm) height was evaluated. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in jump performance (m/s) | The drop jump test on a force mobile platform (AccuPower, AMTI, USA) was used to evaluate jump performance. Jumps were performed while holding the hands of the subject on the hips were requested jump as fast as possible after the drop off from the platform and make sure that the jump is the highest possible. Time to take off (in m/s) was evaluated. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in height (m) | Height (in m) was measured using a Harpenden anthropometer set (Holtain Ltd, UK) | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in skinfold thickness (mm) | Skinfolds thickness (in mm) was measured using a skinfold caliper (Saehan, Korea) at 7 sites: chest, midaxilla, triceps, subscapular, abdominal, suprailiac and thigh. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in body fatness (%) | 7-Site Skinfold Jackson & Pollock protocol was used to estimate body fat (in %) based on measurements of subcutaneous fat. As presented earlier, skinfolds were measured at 7 sites, and further body fatness was calculated as 495/(1.112-(0.00043499*s(sum of 7 skinfolds)+(0.00000055*s*s)-(0.00028826*a(age)))-450 for males and 495/(1.097-(0.00046971*s)+(0.00000056*s*s)-(0.00012828*a))-450 for females. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in physical activity (Step count) | Garmin activity tracker estimated the step count. | 3 days before condition, during 48 or 144 hours of interventions and during 1 week post-condition. (2 weeks) | 
| Change in sleep (h) | Sleep statistics including total hours of sleep and the time spent in different sleep stages were assessed with Garmin activity tracker. | 3 days before condition, during 48 or 144 hours of interventions and during 1 week post-condition. (2 weeks)] | 
| Change in oxygen consumption and carbon dioxide output (ml/min) | Oxygen consumption and carbon dioxide output (in ml/min) every 5 s on a breath-by breath basis using an Oxycon Mobile spirometry system (Oxygen Mobile, Jaeger/ VIASYS Healthcare, Germany) was measured at rest. | Pre-condition, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in capillary blood ketone concentrations (mmol/l) | The capillary blood ketone concentration (in mmol/l) was assessed using an Abbott FreeStyle Optium Neo H blood glucose and ketone monitoring system (Australia) using blood obtained from a finger-prick sample. | Pre-condition, 24, 48 or 144 hours post-condition, and 1 week after recovery | 
| Change in capillary blood glucose concentrations (mmol/L) | The capillary blood glucose concentration (in mmol/L) was assessed using an Abbott FreeStyle Optium Neo H blood glucose and ketone monitoring system (Australia) using blood obtained from a finger-prick sample. | Pre-condition, 24, 48 or 144 hours post-condition, and 1 week after recovery | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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cognition, motor function, metabolism, young adults, fasting, starvation, men, women, calorie deprivation, stress, kynurines, catecholamines, glucose tolerance, energy expenditure, weight loss