Participants

This study was approved by the Institutional Review Board of Osaka City University Graduate School of Medicine (No. 4033) and conformed to the standards of the Declaration of Helsinki. Written informed consent was obtained prior to participation in the study.

Nine active healthy males, who usually perform light-to-moderate exercise a few days per week to maintain their health, volunteered to participate in this study. All participants were nonsmokers, normotensive, and had no overt history of cardiovascular or metabolic diseases. Their age, height, body weight, body mass index, and peak oxygen uptake (V̇O2peak) were 21.1 ± 0.6 years, 172.0 ± 3.9 cm, 65.2 ± 9.6 kg, 22 ± 3 kg∙m−2, and 42.9 ± 5.3 mL∙kg−1∙min−1 (means ± standard deviation), respectively.

Study design and procedures

At least 1 week before the first trial, V̇O2peak was determined under a temperate condition, ambient temperature of ~ 24 °C, and relative humidity of ~ 32% by a graded exercise test with an upright cycle ergometer (828E; Monark, Varberg, Sweden). While the participants kept pedaling at 60 cycles/min, the exercise load was first set at 0 W for 3 min, and then increased by 60 W every 3 min up to 180 W. Above this intensity, it was increased by 30 W every 2 min up to 240 W and then by 15 W every 2 min until the participants could not maintain the rhythm due to exhaustion.

Each participant performed mental stress (MS) and control (CON) trials in a counterbalanced order. The trials were performed at least 5 days apart to avoid any physical or psychological fatigue from winter to spring season in Japan (end of February to April). The participants were instructed to refrain from consuming beverages containing caffeine or alcohol and to avoid strenuous physical activity for at least 24 h before each trial. To ensure that the participants started the trials well-hydrated, they were instructed to drink 500 mL of water before going to bed on the night before the trial and approximately 2 h before arrival on the day of the trial. Additional water was permitted as required. On the day of the trial, the participants arrived at the laboratory in the morning (0930), afternoon (1330), or evening (1530). Two trials were performed at the same time of the day in each subject. The participants ate the same light meal for at least 2 h before arrival in both trials. Upon arrival, the participants emptied their bladders and were weighed without clothing. They then donned short pants. While the participants sat quietly on a stool for 15 min at an ambient temperature of ~ 28 °C and a relative humidity of ~ 33%, an earplug-type thermistor probe was placed into the ear canal to measure ear canal temperature (Tear-c), and skin thermistor probes were placed at four sites on the skin surface to measure skin temperatures. Electrodes were placed on the skin of the chest to trace the electrocardiogram (ECG), and a blood pressure measurement cuff was placed around the upper left arm. A 20-G Teflon-coated catheter (SR-FS2032 or SR-FVP2032P; Terumo, Tokyo, Japan) was inserted into the left antecubital vein for blood sampling. Finally, the participants wore a tube-lined water perfusion suit (F500806; Med-Eng Holdings, Ottawa, Canada) that covered the entire body except for the face, hands, and feet. Participants entered an artificial climatic chamber (TBR-6W2S2L2M; Espec Co., Osaka, Japan) controlled at an ambient temperature of 28 °C and a relative humidity of 33%. They sat quietly on a sofa chair, and the suit was perfused with water maintained at 34 °C with a thermostatic bath (LTB-250α; AS ONE Co., Osaka, Japan) at a flow rate of 1260 mL·min−1. The participants wore face masks for respiratory measurements.

After 20 min, all the measurement signals were confirmed to be in a steady state, and baseline data were collected for 20 min. At baseline, the participants answered psychological questionnaires, a cognitive function test was performed, and expired gas was measured for 5 min. Subsequently, the water temperature in the suit was changed to 10 °C for 10 min and then to 15 °C for up to 85 min for whole-body surface cooling, while the participants rested in the sitting position. This approach was chosen to enable precise control of skin temperature without altering core temperature or ambient conditions and avoid inducing shivering. During cooling, the participants performed the Stroop color-word test for the MS trial or watched a documentary video for the CON trial. After the intervention, participants answered the questionnaire, expired gas was measured for 5 min, and the cognitive function tests were performed again within 20 min while water temperature in the suit was maintained at 15 °C. Participants then removed the water perfusion suits, and moved immediately to an upright cycle ergometer adjacent to the sofa chair to begin the cycling performance test. The test started within 25 min after the control or mental stress intervention. This design allowed us to examine the effects of pre-exercise cold exposure without introducing thermal influences during the performance test. The time interval between initiation of cold-water circulation and exercise onset ranged from 75 to 98 min, primarily due to minor procedural time differences and time required for voiding bladder before exercise. While this duration varied slightly across participants, it remained consistent within individuals across trials. The mean duration of cold exposure prior to exercise was 85.3 ± 5.8 min in the CON trial and 85.8 ± 7.0 min in the MS trial, with no significant difference between conditions (paired t test, P = 0.870). While the participants kept pedaling at 60 cycles/min, the exercise load was first set at 0% (free pedal) for 1 min and increased by 20% every minute up to 80% of V̇O2peak. The participants continued to pedal until they could not maintain their rhythm because of exhaustion. Ratings of preserved exertion (RPE) were measured using Borg’s 15-point scale (Borg 1982) at the start and every 3 min of exercise at 80% V̇O2peak and at exhaustion. TE was evaluated for endurance exercise. The criteria for exhaustion were as follows: subjective exhaustion with RPE of 19 or 20 and pedaling rate < 50 cycles/min. HR and oxygen uptake (V̇O2) was measured throughout the exercise. After the exercise, the participants answered the questionnaire again. 11 mL of blood samples was obtained before and after the intervention, and at the end of the exercise to determine blood chemicals and endocrine hormones.

InterventionMS trial

The Stroop color-word test (Stroop 1935) was used to assess psychological stress as previously reported (Van Cutsem et al. 2017a; Otani et al. 2017). In this task, colored words (‘‘green’’, ‘‘red’’, ‘‘yellow’’, and ‘‘blue’’ shown in Japanese) were indicated one at a time on a monitor, and participants were required to answer the color of the word using a keyboard, ignoring the meaning of the word itself. The trials were arranged in a random sequence, with 25% of the trials being congruent (matched word and color), whereas 75% were incongruent, with all incongruent word-color combinations being equally common. First, black cross text of font size 28 was presented for 1000 ms on a white background. Each color word was then presented on the screen in font size 28 until the participants reacted. Subsequently, the results of the reaction were provided as feedback on correct or incorrect, reaction time, and mean reaction time on a white screen in a font size 18 for 1500 ms before the fixation text was displayed again. Therefore, a new word was presented every 2500 ms plus the response time, providing a total of 500 stimuli per set (the four-color words were presented 125 times each). Two sets were imposed, with a 60-s brake between the sets. This task was performed using E-Prime, version 3.0, Psychology Software Tools (Sharpsburg). The participants were instructed to respond as quickly and accurately as possible. The task lasted 54.2 ± 5.7 min for two sets, including both fixed stimulus display time and participants’ response time.

CON trial

Participants watched a 55-min documentary video (Urban train collection of West Japan Railway, Vicom Co., Ltd., Kurume, Japan) on the same computer screen used for the Stroop color-word test. To exclude the effects of sound, the participants watched the video without sound.

MeasurementsBody temperature

Tear-c was measured using the earplug-type thermistor (LT-2N-13; Gram Co., Saitama, Japan). Tsk was measured using skin thermistors (LT-ST8-12; Gram Co.) at four sites: right side of the chest (Tchest), upper arm (Tarm), thigh (Tthigh), and leg (Tleg). The position of each site was marked and measured to place the thermistors at identical positions in both trials. Temperature data were collected every second throughout the experiment using a computerized data acquisition system (intercross310; Intercross Co., Tokyo, Japan) and stored on a laboratory computer (JAP32301WP; Hewlett-Packard Japan Ltd., Tokyo, Japan). The mean skin temperature (Tsk) was calculated as 0.3 (Tchest + Tarm) + 0.2 (Tthigh + Tleg) (Ramanathan 1964).

Hemodynamics and metabolism

HR was recorded at 1-min intervals using a trace ECG (BSM-7200; Nihon Kohden Co., Tokyo, Japan). Systolic blood pressure and diastolic blood pressure were measured at 5 min intervals with auscultation using a cuff placed on the left upper arm at the heart level (STBP-780, Colin, Komaki, Japan). V̇O2 and carbon dioxide excretion (V̇CO2) were calculated every 20 s (AE-310S; Minato Medical Science, Co., LTD., Osaka, Japan). Respiratory exchange ratio (RER) was calculated as V̇CO2/V̇O2. Energy expenditure was calculated as 3.9 V̇O2 + 1.1 V̇CO2 (Weir 1949). Carbohydrate and lipid oxidation rates were calculated as 4.59 V̇CO2–3.23 V̇O2 and − 1.70 V̇CO2 + 1.70 V̇O2, respectively (Haman et al. 2007).

Blood chemicals and endocrine hormones

A 4.0-mL aliquot of each blood sample was transferred to a heparin-treated tube immediately after sampling. A 1.0-mL sample was used to determine the hematocrit by the micro-centrifuge method, in triplicate. Hematocrit values were corrected using multipliers of 0.96 for trapped plasma and 0.91 for the F cell ratio (Harrison 1985). A 3.0 mL of the sample was centrifuged at 4 °C for 15 min. The separated plasma sample was stored at − 80 °C until used to measure plasma lactate and glucose concentrations (immobilized enzyme method, YSI 2300 STAT PLUS; YSI Inc., Yellow Springs) and total protein concentration (refractometry, ATAGO SPR-T2; Atago Co., Tokyo, Japan). A 7.0-mL aliquot of each blood sample was transferred to a tube containing 1.5 mg·mL−1 EDTA-2Na and centrifuged at 4 °C for 15 min. The separated plasma sample was stored at − 80 °C until used to measure plasma concentrations of catecholamine (adrenaline, [Ad]p, noradrenaline, [Norad]p, and dopamine, [Dopa]p; high-performance liquid chromatography, LSI Med. Co., Tokyo, Japan) and cortisol ([Corti]p; chemiluminescent immunoassay, LSI Med. Co.). The intra-assay coefficients of variation were as follows: 4.08%, 0.85%, and 0.76% for [Ad]p at 0.10, 0.34, and 0.79 ng·mL−1, respectively; 9.34%, 0.81%, and 0.60% for [Norad]p at 0.10, 0.54, and 5.01 ng·mL−1, respectively; 8.96%, 0.99%, and 1.48% for [Dopa]p at 0.07, 0.26, and 0.61 ng·mL−1, respectively; 3.21%, 1.93%, and 1.75% for [Corti]p at 3.93, 15.97, and 35.60 µg·dL−1, respectively.

Psychological questionnaire and cognitive function tests

The Chalder fatigue score (CFS) questionnaire was used to assess subjective fatigue (Chalder et al. 1993), consisting of eight questions about physical symptoms and six questions about mental symptoms. The questions were answered on a 4-point scale (0 = better than usual, 1 = no more than usual, 2 = worse than usual, and 3 = much worse than usual). Total points ranged between 0 and 42. In addition, four questions for fatigued were included from the Brunel Mood Scale (BRUMS) (Terry et al. 2003). The questions were answered on a 5-point scale (0 = not at all, 1 = a little, 2 = moderately, 3 = quite a bit, and 4 = extremely). The total score for fatigue ranged between 0 and 16 points. The modified Flanker task was used as a cognitive function test to confirm the effects of mental fatigue (Weng et al. 2015). The congruency of the flanking targets consists of either the manipulated left or right arrows, resulting in three conditions: congruent (e.g., >>>>> or <<<<<), incongruent (e.g., >><>> or <<><<), and neutral (e.g., ◊◊>◊◊ or ◊◊<◊◊). Each array of arrows was focally presented in black cross text (font size 34) for 1000 ms on a white background with an inter-stimulus interval of 1000 ms. For each task, 40 trials were presented randomly with the left and right target arrows occurring with equal probability, resulting in a total of 120 trials. If the response time was > 1000 ms, it was considered a non-response. The total duration of the task was approximately 4 min. The participants were instructed to respond as quickly and as accurately as possible to the direction of the target (center) arrow while ignoring the two flankers on each side. To assess performance, accuracy, total time, mean reaction time, and number of non-responses were evaluated. This task was performed using E-Prime.

Statistical analysis

Data were analyzed using SigmaPlot version 14 (Systat Software Inc., San Jose, CA). The normality of data was assessed using the Shapiro–Wilk test. While most variables followed a normal distribution, a few did not meet this assumption. For these variables, results are presented as reference values and interpreted with caution. Paired Student’s t test was used for simple comparisons between trials. To account for potential learning effects, trial order was counterbalanced across participants. We confirmed no significant differences in TE (P = 0.302) or cognitive performance test results at pre-intervention between the first and second trials regardless of condition (P = 0.129–0.625), except for Flanker task mean reaction time which showed an enhanced performance in the second trial compared to the first trial (P = 0.016). Two-way repeated-measures analysis of variance (ANOVA) was used to test for significant effects of trials, time, and interaction, with the Brown–Forsythe test applied to confirm equal variances. Subsequent post hoc tests determined significant differences in each pairwise comparison using the Bonferroni test. For variables showing significant between-trial differences at pre-intervention, adjusted values were analyzed using a one-way analysis of covariance (ANCOVA), with the pre-intervention value included as a covariate to control for initial differences. Pearson’s product-moment correlation coefficient was calculated to evaluate the linear relationship between the variables. Effect sizes were calculated and reported using Cohen’s d, where < 0.20 is small, > 0.20 is moderate, and < 0.50 are small, moderate, and large, respectively (Cohen 1988). Significance was set a priori at P < 0.05, and moderate-to-large effect sizes were considered meaningful. Values are expressed as the means ± standard deviation of the mean (SD).