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

Body mass and composition (in kg) was evaluated using Tanita Body Composition Analyzer (Japan).

The body mass index (in kg/m2) was defined as the body mass divided by the square of the body height.

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.

Resting energy expenditure (REE) using Weir equation modified Weir equation: REE (kcal/day)=[3.941(oxygen consumption) + 1.106(carbon dioxide output)] x 1440

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).

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).

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).

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.

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).

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.

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).

Heart rate (in bpm) was recorded using a heart rate sensor with a chest strap (Polar, Finland) in laying position at rest.

Resting systolic and diastolic blood pressure (in mmHg) was measured using a digital electronic blood pressure monitor (Microlife, Switzerland)

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".

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".

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.

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).

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).

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).

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).

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).

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).

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).

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).

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).

The venous plasma malondialdehyde concentrations (in nmol/l) were measured using a solid phase nzyme-linked immunosorbent assay (Cat. No. E1371Hu, Bioassay Technology Laboratory, Shangai, China) and a Spark multimode microplate reader (Tecan, Austria).

The venous plasma total antioxidant capacitys (in mmol/l) were measured colorimetrically with an assay kit (Cat. No.E-BC-K271-M, Elabscience Biotechnology Inc, Houston, USA) and using a Spark multimode microplate reader (Tecan, Austria).

The venous glucose concentration (in mmol/l) was measured in venous blood samples using a Glucocard X-mini plus glucose analyser (Arkray, Japan).

An oral glucose insulin sensitivity [OGIS] index derived from an oral glucose tolerance test was calculated.

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.

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).

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).

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).

The Grooved Pegboard was used to evaluate the ability to coordinate the fingers and manipulate objects promptly in time twice (in sec).

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.

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.

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.

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.

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.

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.

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.

Tibial muscles electromyographic (EMG) amplitude (in mV) parameters of muscular activity were measured using surface EMG (Biometrics, UK) thorough neuromuscular function assessment.

Tibial muscles electromyographic (EMG) frequency (in Hz) parameters of muscular activity were measured using surface EMG (Biometrics, UK) thorough neuromuscular function assessment.

Isometric and isokinetic voluntary torques (in Nm) of the ankle plantar flexion/dorsiflexion muscles were measured using an isokinetic dynamometer (Biodex Medical Systems, USA).

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.

The contraction and half-relaxation time (in ms) were measured in resting TT100 contractions.

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.

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.

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.

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.

Height (in m) was measured using a Harpenden anthropometer set (Holtain Ltd, UK)

Skinfolds thickness (in mm) was measured using a skinfold caliper (Saehan, Korea) at 7 sites: chest, midaxilla, triceps, subscapular, abdominal, suprailiac and thigh.

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.

Garmin activity tracker estimated the step count.

Sleep statistics including total hours of sleep and the time spent in different sleep stages were assessed with Garmin activity tracker.

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.

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.

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.