Differences in stress response between two altitudes assessed by salivary cortisol levels within circadian rhythms in long-distance runners

Participants

This study was conducted in accordance to the Declaration of Helsinki, and the protocol was approved by the ethics committee of the Gunma University Graduate School of Medicine (Approval number HS2018-140). All the study participants provided written informed consent before being included in the study.

We enrolled 12 Japanese elite female long-distance runners. All of them lived in the same dormitory before the first training camp and stayed in the same hotels during camps. Their living conditions, such as wakeup time, meal time, bedtime, and meal content, were standardized before and during the training camps16. Figure 1A presents the schedules of the pre-camp and the two training camps. This altitude training was a study without invasive interventions, because it was usually conducted to improve the condition of the runners. The runners lived and trained near sea level (150 m) before the first training camp. Then, for the first training camp simulating the higher-altitude camp at low altitudes, they lived at 1800 m and trained at 1700 m twice a day, morning and afternoon, for 7 days. Afterwards, for the second training camp simulating the lower-altitude camp at low altitudes, they lived at 1550 m and trained at 1300 m twice a day, morning and afternoon, for 7 days. They then returned to near sea level. We sequentially collected saliva from these runners on the last 2 days during each camp which involved different exercise intensities in the morning and afternoon, modified as previously described16. Figure 1B details the relation between runner’s altitudes and saliva collection times during 2 consecutive days during each training camp. On both days, saliva samples were collected at eight time points: upon waking (05:00), before morning exercise (06:00), after morning exercise (07:00), before breakfast (08:00), before lunch ( 12:00), before afternoon exercise (15:00), after afternoon exercise (16:00), and before dinner (18:00), as described previously16. On each training day at both camps, no differences were observed in the meteorological conditions; the temperature was around 20 ° C and the relative humidity was 50–60% with fine weather. The runners were subjected to the following exercise program in the higher-altitude camp: 40-min fixed running in the morning and 50-min fixed running in the afternoon on day 1 (Higher-day 1); 8000-m fixed-distance running in the morning and uphill interval training with 8 sets of 200-m fast uphill running and light jogging in the evening on day 2 (Higher-day 2). The runners were subjected to the following exercise program in the lower-altitude camp: 50-min fixed running in the morning and 60-min fixed running in the afternoon on day 1 (Lower-day 1); 8000-m fixed-distance running in the morning and uphill interval training with 5 sets of 200-m fast uphill running and light jogging in the evening on day 2 (Lower-day 2). The runners drank enough water to prevent dehydration during these trainings.

Figure 1

The study design of the first and second training camps of 12 female long-distance runners. The altitudes at which the runners lived and trained during the two training camps and pre- and post-camp (TO). The schema of altitudes and saliva sampling time in runners on the last 2 days during each training camps (B.). The downwards arrows denote the saliva sampling from the runners, and the tips of the arrows denote the altitudes at the time of sampling at both camps. Higher-day 1, day 1 at the higher-altitude camp; Higher-day 2, day 2 at the higher-altitude camp; Lower-day 1, day 1 at the lower-altitude camp; Lower-day 2, day 2 at the lower-altitude camp.

Physical examinations

The participants were weighed, and their body mass indexes were calculated as the weight divided by the squared height (kg / m2). After conducting interviews, no runners were found to use any medications or supplements. The runners used the Apple Watch Series 3 (Apple Japan Inc., Tokyo, Japan) during the camps. This allowed for the measurement of maximum pulse rate during each exercise and resting pulse rate at awakening and before dinner. The distance and duration of running during each exercise session was measured, and the running velocity was calculated as the distance divided by the duration (m / min)15. The Borg Rating of Perceived Exertion (RPE) scale20 was utilized to measure the runner’s subjective exertion, breathlessness and fatigue after exercise. The runner’s RPE was scored using a scale ranging from 6 to 20 and used in the analysis as a Borg scale score.

Saliva collections and measurements of salivary cortisol concentrations

Sample collections and salivary cortisol concentration measurements were performed according to a previous study16. The runners were not allowed to brush their teeth, chew gum, or consume any food or drink except water, 15 min before the sample collection. Saliva samples were collected using Salivette® cotton swabs (Sarstedt, Nümbrecht, Germany), centrifuged (1,500 × g) at 4 ° C for 10 min, then immediately stored at – 80 ° C until analysis. ECLIA measurements of salivary cortisol concentrations were performed using the Elecsys Cortisol II on the Cobas 8000 system (Roche Diagnostics KK, Tokyo, Japan)15.16. The intra- and inter-assay coefficients of variation for salivary cortisol were 4.1% and 4.6%, respectively. The rate of change in the salivary cortisol concentration by exercise was calculated as the salivary cortisol concentration after exercise divided by the salivary cortisol concentration before exercise (%)16.

Statistical analysis

The results of each measurement are expressed as the median values ​​and corresponding 25th – 75th percentile ranges. The Wilcoxon signed-rank test was utilized to identify statistically significant differences in variables between two different time points. TOp value of <0.05 was considered statistically significant. All statistical analyzes were performed using SPSS Statistics, version 26.0 (IBM Corp., Armonk, NY, USA).

Ethics approval and consent to participate

Written informed consent was obtained from all participants. This study was approved by the ethics committee of Gunma University Graduate School of Medicine (Approval number HS2018-140). All measurements were carried out by trained athletes and in accordance with the Declaration of Helsinki.

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