Study Design and Setting
To investigate the effects of high-intensity resistance exercise on CAR, this study consisted of strength testing and four consecutive days of experimental session (Figure 1). The first visit (strength testing) was used for screening and measuring their one-repetition maximum (1RM). The next four days were conducted experimental session that continued as follows: two days of sedentary control sessions (Day 1 and Day 2), a high-intensity resistance exercise session until exhaustion (HIRE; Day 3), and a recovery session considering the effect of delayed onset muscle soreness which occurs 8 to 72 hours after exercise (Recovery; Day 4) (18). Strength testing and experimental sessions were separated by at least 72 hours. Since bench press exercises have been commonly used among various trained individuals and the load can be easily controlled to ensure safety (19), a bench press exercise was selected as a high-intensity resistance exercise session in this study. In addition, we first examined the effect of intensity aimed at muscle hypertrophy (75% of 1 RM), which was observed to increase greater acute cortisol levels in previous studies (20, 21). The CAR was measured during the day after each session, and each response was compared. Additionally, as CAR has high intra-individual stability, whereas high inter-individual variability (12), we also analyzed focusing on the inter-individual variability.
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Before the experiment, all participants were provided with an oral and written informed consent. Fourteen healthy male university students with at least 1 year of resistance exercise experience participated in this study (mean ± SD; 21.9 ± 0.9 years, 171.2 ± 5.8 cm, 68.3 ± 6.0 kg, 23.3 ± 1.4 BMI, 88.08 ± 17.8 1 kg bench press 1RM). Exclusion criteria included a history of hormonal disorders, mental illness, smoking, a diet chronically low in carbohydrates, use of anabolic steroids, or chronic nonsteroidal anti-inflammatory drug use. One participant was excluded since he was affected by other exercises during the experimental period. Therefore, 13 participants’ data were used for the further analyses. This research was approved by the Ethics Committee at Osaka University of Health and Sport Sciences (approval number: 20-19).
At least 72 hours prior to the experimental session, each participant had their 1 RM determined on the bench press exercise. The 1 RM tests were conducted using the National Strength and Conditioning Association protocol for 1 RM testing (22). The protocol required the participants to progressively increase the resistance across several trials until the 1 RM was achieved. During the trials, the participants were asked to raise to maximum speed for all the trials. The movement velocity (MV) was measured on each repetition to determine the 1 RM based on the load-velocity relationship. The MV showed on the last successful repetition was defined as the movement velocity threshold (MVT) (23), which was used to evaluate the training load during the HIRE session.
On Day 1 and Day 2, a sedentary control session (20 minutes) was conducted each day to measure their baseline values. On Day 3 (HIRE session), the participants performed bench press exercises with (a) 20 kg for 10 repetitions and (b) 40% for 10 repetitions as a warm-up exercise. After the warm-up, they performed 10 repetitions of bench press exercises at 75%. Following a 90-second rest, they performed 5-8 sets again until exhaustion. On Day 4, a Recovery session without any physical activity for a day was conducted for an additional day after the HIRE session.
As shown in Figure 1, measurements for the control and the HIRE sessions were taken based on previous research (16); before (Pre) and immediately after (Post-0) the experimental session, during the recovery period of the session (every 10 min for 30 min; Post-10, Post-20, and Post-30), the night after returning home from the session (9 pm, 11 pm; Recovery 1 and Recovery 2), and upon waking up the day after the session. The Recovery session of Day 4 was conducted only to measure upon waking up the next day. All participants completed the sessions at the same time of the day (6 pm ± 30 min) to control for biological variation. They were asked to eat similar meals during the experiment and to refrain from eating at least 4 hours before the experiment and no later than 8 pm after the experiment. In addition, the participants were required to refrain from exercise, caffeine, and alcohol during the experiment.
Evaluation of bench press exercise
To evaluate whether the HIRE session was performed to exhaustion, MV and the rating of perceived exertion (RPE) of the upper limb were measured. MV was measured during each repetition of the HIRE session using accelerometers (PUSH Band 2.0: PUSH Inc., Canada). The RPE of the upper limb was determined using Borg's 6-20 points rating of perceived exertion scale, a validated Japanese questionnaire (24). The HIRE session was considered valid and reliable if the participants met three of the following criteria: 1) MV at the last repetition showed equal to or lower than MVT (23), 2) the RPE of the upper limb rating was of 19 or greater, and 3) participants were unable to lift the bench press by themselves.
Saliva samples were collected (Figure 1) by the passive drool techniques. Prior to the experiment, the participants were informed of the guidelines for saliva collection. Participants collected saliva in their mouths for 2 minutes before pouring it into a tube using a straw. To avoid saliva dilution, no mouthwash was utilized. Participants were asked not to drink, wash their teeth, or bathe for 1 hour before saliva collection for Recovery 1 and 2. During the wakeup phase, they were also not allowed to eat, drink, brush their teeth, engage in vigorous physical activity, or return to sleep, but they were allowed to engage in other normal morning activities. On a Google form, the participants were asked to report their sleeping time, waking time, saliva collection time, and sleeping status. Participants were asked to preserve saliva samples in a commercial freezer at minus −4°C for 48 hours before submitting them to the laboratory. To precipitate particulate particles, the saliva samples were centrifuged at room temperature for 5 minutes at 3000g and kept at minus −80°C until analysis. Following that, cortisol concentrations were determined using an enzyme-linked immunosorbent test (Cortisol ELISA Kit (RE52611), IBL, Japan). The magnitude of change (CARc), the relative change (CAR%), and the area under the curve were determined in two ways from the samples collected upon awakening: relative to the ground (compared to a "zero" concentration; AUCg) and relative to the increase (related to the first concentration; AUCi) (13, 9).
Factors influencing the CAR
To explore the factors that changed the CAR resulting from the HIRE session, physical conditions (perceived physical fatigue and muscle soreness of the upper limb) were assessed using a visual analogue scale (VAS). In addition to excluding confounding factors, factors that could influence CAR were measured upon awakening. The degree of stress and sleep quality were evaluated by VAS. The psychological condition was assessed with a total mood disturbance (TMD) score calculated from the short form of the Profile of Moods States Second Edition (POMS2-S), which has been translated into Japanese (25). Participant graded a set of 35 items related to the mood on a Likert scale from 0 (not at all) to 4 (extremely) to such question "How do you feel at this moment?" to assess 7 dimensions: Anger-Hostility (AH), Confusion-Bewilderment (CB), Depression-Dejection (DD), Fatigue-Inertia (FI), Tension-Anxiety (TA), Vigor-Activity (VA), and Friendliness (F).
Data were expressed as mean ± standard error. A paired t-test was used to compare the MVT and MV at the last repetition of the HIRE session. Two-way repeated-measures ANOVA tests were conducted to analyze the changes in cortisol concentrations on the day of the experimental session (session  × sampling point ) and CAR on the day after the session (session  × sampling point  and session  × sampling point ). One-way repeated measure ANOVAs were used to analyze the difference between experimental sessions in CAR data (CARc, CAR%, AUCg, and AUCi), VAS (physical fatigue, muscle soreness, stress, and sleep quality), the TMD score during awakening, sleeping hours, and awakening time. Mauchly's test was used to assess the sphericity assumption. If the assumption was violated, Greenhouse-Geisser epsilon values were used to adjust the degrees of freedom. Bonferroni corrections were used for post-hoc tests. Estimate of effect size was quantified partial eta squared (ηp2), where ηp2 = 0.01, 0.06, and 0.14 were estimated for a small, moderate, and large effect, respectively (26). We also conducted correlation analyses (Pearson correlation) to assess the relationship between the rate of change in CAR data (CARc, CAR%, AUCg, and AUCi) and the amount of change in perceived physical condition data (physical fatigue and muscle soreness).
The level of significance was set to α ≤ .05. In addition, a more liberal α value of ≤ .10 was used to determine marginal significance to avoid Type Ⅱ error (27). All statistical analyses were performed using SPSS version 27.0 (IBM, Japan).