Effect of a 16-day Hot and Cold Acclimation on Adaptive Responses and Health-related Indicators

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

Skinfolds thickness (in mm) will be measured using a skinfold caliper (Saehan, Korea) at 10 sites and the mean subcutaneous fat thickness will be calculated.

The body mass index (in kg/m^2) will be defined as the body mass divided by the square of the body height.

The participant's body surface area (BSA) (in m^2) will be estimated using the following equations: BSA = 128.1 ×weight^0.44 × height^0.60 for males and BSA = 147.4 ×weight^0.47 × height^0.55 for females.

Oxygen consumption (VO2) and carbon dioxide (VCO2) output will be measured using Cortex METALYZR® 3B, Leipcig, Germany), and the respiratory quotient (RQ = VCO2 / VO2) will be computed to determine substrate utilisation. The RQ values for fat is assumed as 0.7, for protein is assumed as 0.8 and for carbohydrate isassumed as 1.0.

Carbohydrate and fat oxidation (in g/day) will be measured using Cortex METALYZR® 3B, Leipcig, Germany).

Ventilation (in l/min) will be measured using Cortex METALYZR® 3B, Leipcig, Germany).

Breathing frequency (in t/min) will be measured using Cortex METALYZR® 3B, Leipcig, Germany).

The resting energy expenditure (REE; in kcal/day) will be computed using the following equation: REE = [3.941 × (VO2) + 1.106 × (VCO2)] × 1440

The metabolic heat production (MHP; in W) will be computed using the equation: MHP = (281.65 + 80.65 × RQ) × VO2.

Heart rate (in bpm) will be recorded using a heart rate sensor with a chest strap (Polar, Finland) or Physioflow hemodynamic monitor (France).

Systolic and diastolic blood pressure (in mmHg) will be measured using PROVIEW 10 Compact Patient Monitor (Germany).

The mean arterial pressure (MAP; in mmHg) will be estimated using the formula: MAP = DP + 1/3(SP - DP) or MAP = DP + 1/3(SP - DP), where SP is systolic bood pressure and DP is diastolic blood pressure.

R-R intervals (in ms) in supine resting will be recorded using a Polar heart ratesensor (Finland) and simultaneously transferred to Polar Pro Trainer 5 software (Finland)

Heart rate variability data will be analyzed using Kubios heart rate 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 will be assessed (in ms).

Heart rate variability data will be analyzed using Kubios heart rate 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) will be assessed and logarithmically transformed (Ln) to correct the skewness of distribution.

Heart rate variability data will be analyzed using Kubios heart rate variability analysis software (Finland). In the frequency domain that measures the more specific contribution of the autonomic nervous system branch, low-frequency (LF; estimates sympathetic and parasympathetic activity) and high-frequency (HF; estimates parasympathetic activity) powers in absolute units (in m^2) will be assessed.

In the frequency domain that measures the more specific contribution of the autonomic nervous system branch, low-frequency (LF; estimates sympathetic and parasympathetic activity) and high-frequency (HF; estimates parasympathetic activity) powers will be assessed and logarithmically transformed (Ln) to correct the skewness of distribution.

Stroke volume (in ml) will be recorded using Physioflow hemodynamic monitor (France).

Stroke volume index (in ml/m^2) will be recorded using Physioflow hemodynamic monitor (France).

Cardiac output (in l/min/m^2) will be recorded using Physioflow hemodynamic monitor (France).

Cardiac output (in l/min) will be recorded using Physioflow hemodynamic monitor (France).

Contractility index will be recorded using Physioflow hemodynamic monitor (France).

Ventricular ejection time (in ms) will be recorded using Physioflow hemodynamic monitor (France).

Ejection fraction (in percent) will be recorded using Physioflow hemodynamic monitor (France).

End diastolic volume (in ml) will be recorded using Physioflow hemodynamic monitor (France).

Systemic vascular resistance (in dyn.s/cm5.m^2) will be recorded using Physioflow hemodynamic monitor (France).

Cardiac work index (in kg.m/m^2) will be recorded using Physioflow hemodynamic monitor (France).

Early diastolic filling ratio (in percent) will be recorded using Physioflow hemodynamic monitor (France).

The saliva samples will be collected to measure testosterone level (in µg/dl) using an ELISA kits and a Spark multimode microplate reader (Tecan, Austria).

The venous 17beta-estradiol and progesterone (in pg/mL), follicle stimulating hormone and luteinizing hormone will be measured using an ELISA kits and a Spark multimode microplate reader (Tecan, Austria).

The saliva samples will be collected to measure cortisol level (in µg/dl) using an ELISA kits and a Spark multimode microplate reader (Tecan, Austria)..

The venous tumor necrosis factor alpha, interleukin-6 and IL-1 beta (in pg/ml) will be measured using an ELISA kits and a Spark multimode microplate reader (Tecan, Austria).

Complete blood count with 5 different white blood count components (absolute neutrophils, lymphocytes, monocytes, eosinophils, basophils) analysis (in 10^9/L) will be performed using an automated Mythic 60 hematology analyzer (Switzerland).

Complete blood count with 5 different white blood count components (absolute neutrophils, lymphocytes, monocytes, eosinophils, basophils) analysis (in percent) will be performed using an automated Mythic 60 hematology analyzer (Switzerland).

Blood samples will be collected to measure lipid profile (in mmol/l) (total cholesterol, high density and low density cholesterol, triglycerides) using a CardioChek PA analyzer (USA).

The venous adrenaline and noradrenaline concentrations (in ng/ ml) will be measured using enzyme-linked immunosorbent assay kits and a Spark multimode microplate reader (Tecan, Austria).

The venous insulin concentrations (in μIU/ml) will be measured using enzyme-linked immunosorbent assay kits and a Spark multimode microplate reader (Tecan, Austria)

The glucose concentration (in mmol/l) will be measured using a On-call GK dual meter (Acon Laboratories, USA).

The level of anxiety and depression (in points) will be defined using Hospital Anxiety and Depression scale. Scale scores range from 0 to 21, with higher scores indicating more severe symptoms.

An ultra-performance liquid chromatography-tandem mass spectrometry system (UPLC-MS/MS) will be used 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 uses a Xevo TQ-XS triple quadrupole mass spectrometer (Waters) with a Z-spray electrospray interface, and the system operates in electrospray positive multiple reaction monitoring mode.

Rectal temperature (in °C) will be measured using a thermocouple (Rectal Probe, Ellab, Denmark) inserted to a depth of 12 cm past the anal sphincter, skin temperature (in °C) will be measured with thermistors (Skin/Surface Probe, DM852, Ellab) at four sites: supraclavicular, back, thigh, and forearm, and right lateral gastrocnemius muscle temperature (in °C) will be measured using a needle microprobe (MKA; Ellab).

A physiological strain index (PSI) will be used to indicate heat strain. PSI = 5 x (Tret - Tre0) x (39.5 - Tre0)^-1+ 5 x (HRt - HR0) x (180 - HR0)^-1, where rectal temperature (Tre) t and heart rate (HR) t are simultaneous measurements taken at the end of the heat exposure and Tre0 and HR0 are the initial measurements.

A cold strain index (CSI) will be used to indicate cold strain. CSI = 6.67 x (Tre t - Tre 0) x (35 - Tre 0)^-1 + 3.33 x (Tsk t - Tsk 0) x (20 - Tsk 0)^-1, where rectal temperature (Tre) 0 and skin temperature (Tsk) 0 are initial measurements and Tre t and Tsk t are simultaneous measurements taken at the end of the cold exposure.

Root mean square (RMS) amplitude (in mV) of pectoralis major muscle indicating shivering intensity will be measured using a surface EMG (Biometrics, UK).

The pain sensation will be evaluated using numeric pain scale ranging from 0 (no pain) to 10 (worst possible pain).

The thermal sensation will be evaluated using 9-point scale. The rating of thermal sensation range from 1 (very cold) to 9 (very hot), with 5 being neutral.

The shivering/sweating rate will be evaluated using 7-point scale. The rating of shivering/sweating range from 1 (heavily sweating) to 7 (vigorously shivering), with 4 being neutral.

The thermal comfort will be evaluated using 4-point scale. The rating of thermal comfort range from 0 (neutral) to 3 (very uncomfortable).

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

MotionWatch 8 actigraph will estimate the time spent in sedentary, light, moderate, or vigorous activity

Sleep statistics (in h) including total hours of sleep and the time spent in different sleep stages will be assessed with MotionWatch 8 actigraph.

Oxygen saturation (in percent) will be measured using Proview 10 Compact Patient Monitor (Germany).