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Differences in stress response 
between two altitudes assessed 
by salivary cortisol levels 
within circadian rhythms 
in long‑distance runners
Katsuhiko Tsunekawa*, Kazumi Ushiki, Larasati Martha, Asuka Nakazawa, Rika Hasegawa, 
Risa Shimizu, Nozomi Shimoda, Akihiro Yoshida, Kiyomi Nakajima, Takao Kimura & 
Masami Murakami
There are conflicting reports regarding the efficacy of cortisol as a stress marker in altitude training 
due to the influence of the circadian rhythm. This study aimed to verify whether the automated 
measurement of salivary cortisol concentration via sequential sampling could detect the differences 
in exercise stress between two altitudes. We enrolled 12 elite female long‑distance runners living 
near sea level. For the first higher‑altitude camp, the runners lived at 1800 m and trained at 1700 m 
for 7 days. For the second lower‑altitude camp, they lived at 1550 m and trained at 1300 m for 
7 days. Their saliva was sequentially collected on the last 2 days during each camp which involved 
different intensity exercises in the morning and afternoon. The salivary cortisol concentrations were 
measured using electrochemiluminescence immunoassay. Before dinner, the basal salivary cortisol 
concentrations were significantly higher in the higher‑altitude camp. The rate of change in the salivary 
cortisol concentration during the morning exercise was significantly higher in the higher‑altitude camp 
than in lower‑altitude camp (p = 0.028) despite the same exercise programs and intensities. Salivary 
cortisol level measurements during the athletes’ circadian rhythms could detect the differences in 
acclimatization and exercise stress between two altitudes.
Abbreviations
ACTH 
 Adrenocorticotropic hormone
ECLIA 
 Electrochemiluminescence immunoassay
ELISA 
 Enzyme-linked immunosorbent assay
RPE 
 Rating of perceived exertion
SpO2 
 Oxygen saturation
VO2 max 
 Maximal oxygen consumption
Elite athletes in various sports often train at high altitudes to improve their performance when they return to 
lower altitudes. At high altitudes with low atmospheric pressure and low oxygen concentrations, the amount 
of red blood cells and their oxygen-carrying capacity are enhanced by an increase in circulating erythropoietin 
concentration due to hypoxia-inducible  factors1. However, a hypoxia during high-altitude training causes exces-
sive stress and decreases an athletes’ performance, resulting in poor  acclimatization2. Therefore, monitoring of 
physical and psychological stress during exercise training at high-altitude camps may help assess maladaptation.
Cortisol is an important biomarker that is secreted into the circulating plasma from the adrenal cortex via 
the hypothalamus–pituitary axis as an acute response to stress including  exercise3. Serum cortisol concentra-
tions are increased by moderate- to high-intensity exercise, but not by low-intensity exercise at less than 40% of 
athletes’ maximal oxygen consumptions  (VO2 max)4,5. However, there have been conflicting reports regarding the 
efficacy of cortisol as a stress marker during high-altitude  training6–11, which may be due to the influence of the 
OPEN
Department of Clinical Laboratory Medicine, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi 
Maebashi, Gunma 371-8511, Japan. *email: ktsune@gunma-u.ac.jp
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circadian rhythm. Changes in the serum cortisol concentrations resulting from exercise are greater in the evening 
than in the morning because of cortisol circadian  rhythm12. Thus, to accurately evaluate exercise-induced stress 
using cortisol, it should be measured throughout day. To achieve this, continuous sampling and more efficient 
cortisol measurement are needed. Serum cortisol concentrations are measured as total hormone conjugated to 
corticosteroid-binding globulin, whereas salivary cortisol concentrations are measured as free hormones inde-
pendent of salivary flow  rates13. Saliva is also advantageous to its ease of collection in the absence of medical 
professional staff and without the stress of  venipuncture14. Conventionally, the salivary cortisol concentration has 
been manually measured using an enzyme-linked immunosorbent assay (ELISA), which this makes it difficult to 
measure a large number of samples compared with automated methods. Recently, the automated electrochemi-
luminescence immunoassay (ECLIA) used for serum cortisol concentration measurement was applied to saliva, 
and the salivary cortisol concentrations measured by ECLIA showed a significantly positive correlation with those 
measured by liquid chromatography-tandem mass  spectrometry15 and with conventional  ELISA16. Moreover, 
we reported that sequential saliva collection and automated ECLIA-based salivary cortisol measurements could 
detect the exercise-induced stress within the circadian rhythm in female long-distance  runners16. In this study, 
the differences in the rate of change in salivary cortisol concentrations resulting from various exercise intensities 
could be compared at the same time on different days, even in the early morning.
Training at altitudes of 500–2000 m, defined as low altitude, causes less stress on athletes than training at 
moderate (2000–3000 m) and high (3000–5500 m)  altitudes17. In contrast,  VO2 max of endurance-trained athletes 
decreased significantly beginning at 300 m above sea level and continued to decrease linearly by approximately 
7% for every 1000 m  ascended18,19. While these reports suggest that there may be differences in athletes’ stress 
responses at different altitudes, even at low altitudes, few biomarkers have ever detected this difference. We 
hypothesized that the automated measurement of salivary cortisol concentrations throughout the athletes’ cir-
cadian rhythms via sequential saliva sampling would enable the assessment of the differences in stress induced 
by training camps at different altitudes, including those at low altitudes. If proven, this method can help in the 
prevention of excessive stress, and foster the development of exercise programs in several altitude environments 
for athletes. The present study expands on our previous study by verifying whether cortisol concentration meas-
urement via continuous saliva collection during the circadian rhythms could adequately detect the differences in 
acclimatization and stress responses resulting from exercise at different altitudes in female long-distance runners.
Methods
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 simulat-
ing 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 morn-
ing 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.
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, breath-
lessness 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.
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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 measure-
ments of salivary cortisol concentrations were performed using the Elecsys Cortisol II on the Cobas 8000 sys-
tem (Roche Diagnostics K.K, 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 correspond-
ing 25th–75th percentile ranges. The Wilcoxon signed-rank test was utilized to identify statistically significant 
differences in variables between two different time points. A p value of < 0.05 was considered statistically signifi-
cant. All statistical analyses 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 par-
ticipants. 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.
Results
Running intensity of each exercise program. 
Table 1 presents the characteristics of the participating 
female long-distance runners, whereas Table 2 presents the different running intensities of each exercise pro-
gram during the two training camps. Because the exercise programs on days 1 and 2 were similar between the 
two camps, the exercise intensities of each program were compared. During the morning exercise on day 1, at 
the higher-altitude camp, the running velocity was significantly higher (p = 0.015) and the running distance and 
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 (A). 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.
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Borg scale scores were significantly lower (running distance, p = 0.029; Borg scale score, p = 0.047) than those at 
the lower-altitude camp. During the afternoon exercise on day 1, the running velocity was significantly higher 
at the higher-altitude camp (p = 0.003), but no differences were observed in other parameters between the two 
camps. During the morning exercise on day 2, the maximum pulse rate was significantly lower at the higher-alti-
tude camp (p = 0.029), but no differences were observed in other parameters between the two camps. However, 
during the afternoon exercise on day 2, the running distance, Borg scale score, and maximum pulse rate were 
significantly higher at the higher-altitude camp (running distance, p = 0.002; Borg scale score, p = 0.005; maxi-
mum pulse rate, p = 0.036). When comparing the exercise intensities between the morning and afternoon on day 
1, the running distance, running velocity, and Borg scale score were significantly higher during the afternoon 
exercise at the higher-altitude camp (running distance, p = 0.002; running velocity, p = 0.002; Borg scale score, 
p = 0.010), whereas the running distance was significantly higher during the afternoon exercise at the lower-
altitude camp (p = 0.004). On day 2, the running distance and Borg scale score were significantly higher and the 
running velocity was significant lower during the afternoon exercise compared with those during the morning 
exercise at the higher-altitude camp (running distance, p = 0.002; Borg scale score, p = 0.004; running velocity, 
p = 0.002), whereas the running distance and velocity were significantly lower during the afternoon exercise at 
the lower-altitude camp (running distance, p = 0.002; running velocity, p = 0.003).
Changes in the salivary cortisol concentrations in response to exercise within the circadian 
rhythms in each camp. 
Figure 2 presents the changes in the salivary cortisol concentrations in response 
to exercise during the last 2 days at both camps. The salivary cortisol concentrations peaked after waking and 
promptly decreased on both days at both camps. Within these circadian rhythms, the salivary cortisol concentra-
tions significantly decreased after the morning exercise on both days at both camps but significantly increased 
after the afternoon exercise on 2 days at only the higher-altitude camp. These concentrations reached their low-
est levels before dinner on both days at both camps. Table 3 presents that the differences in the resting pulse 
rates and salivary cortisol concentrations between the higher- and lower-altitude camps. The resting pulse rate 
before dinner was significantly higher on day 2 at the higher-altitude camp than on day 2 at the lower-altitude 
Table 1.  Characteristics of female long-distance runners. Data are expressed as median (25th–75th 
percentile).
Characteristics
Value
Number
12
Age (year)
23.5 (19.5–26.0)
Height (cm)
160.0 (155.5–164.5)
Weight (kg)
45.5 (41.0–47.5)
Body mass index (kg/m2)
17.4 (17.0–17.9)
Table 2.  Running intensities of the exercise programs performed by female long-distance runners during the 
two camps. Data are expressed as median (25th–75th percentile). *p < 0.05 and **p < 0.01 comparing variables 
between the higher- and lower-altitude camps using the Wilcoxon signed-rank test. pday1 morning exercise vs. 
afternoon exercise on day 1 using the Wilcoxon signed-rank test. pday2 morning exercise vs. afternoon exercise 
on day 2 using the Wilcoxon signed-rank test.
Altitude camp
Day 1
pday1
Day 2
pday2
Morning exercise
Afternoon exercise
Morning exercise
Afternoon 
exercise
Exercise program
Higher
40-min fixed 
running
50-min fixed 
running
8000-m fixed 
running
Uphill interval 
training
Lower
50-min fixed 
running
60-min fixed 
running
8000-m fixed 
running
Uphill interval 
training
Running distance 
(m)
Higher
9700 (9250–
10,150)*
12,790 (12,300–
13,450)
0.002
8000 (8000–8000)
12,000 (9800–
14,000)**
0.002
Lower
11,585 (10,700–
12,000)
13,145 (12,445–
14,000)
0.004
8000 (8000–8000)
4700 (4200–5250)
0.002
Running velocity 
(m/min)
Higher
242.5 (224.8–
247.7)*
252.0 (246.0–
263.0)*
0.002
235.3 (228.6–
238.9)
178.0 (153.9–
215.4)
0.002
Lower
224.2 (205.0–
236.8)
219.1 (207.5–
233.3)
0.477
235.3 (228.6–
236.5)
168.0 (125.0–
213.8)
0.003
Borg scale score
Higher
13.0 (12.0–15.0)*
15.0 (13.0–15.5)
0.010
13.0 (12.0–15.0)
16.0 (14.0–17.5)**
0.004
Lower
14.0 (13.0–15.5)
13.5 (12.0–15.0)
0.119
13.0 (12.0–15.0)
13.0 (12.5–15.0)
0.524
Maximum pulse 
rate (beat/min)
Higher
175 (159–189)
182 (159–201)
0.182
170 (152–184)*
179 (165–192)*
0.167
Lower
168 (153–198)
168 (158–181)
0.937
196 (164–210)
163 (154–187)
0.050
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Figure 2.  Changes in salivary cortisol concentrations in response to each exercise within the circadian rhythm 
on 2 consecutive days during the higher-altitude camp (A) and lower-altitude camp (B). The white box plots 
denote the cortisol concentration at the higher-altitude camp, whereas the gray box plots represent those at the 
lower-altitude camp. The gray dot squares denote the time of exercise at each camp. The significant differences 
between two time points of each exercise for runners were analyzed using the Wilcoxon signed-rank test. 
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.
Table 3.  Comparison of the variables between the higher- and lower-altitude camps in runners. Data are 
expressed as median (25th–75th percentile). *p < 0.05 and **p < 0.01 comparing variables with day 1 at the 
lower-altitude camp using the Wilcoxon signed-rank test. † p < 0.05 and ††p < 0.01 comparing variables with day 
2 at the lower-altitude camp using the Wilcoxon signed-rank test.
Higher-altitude camp
Lower-altitude camp
Day 1
Day 2
Day 1
Day 2
Resting pulse rate at awakening (beat/min)
50 (46–55)
49 (44–52)
48 (44–52)
50 (42–57)
Resting pulse rate before dinner (beat/min)
65 (57–70)
65 (57–71)†
56 (52–68)
54 (48–63)
Salivary cortisol at awakening (μg/dL)
0.41 (0.37–0.45)†
0.36 (0.31–0.42)†
0.40 (0.33–0.43)
0.43 (0.39–0.50)
Salivary cortisol at peak(μg/dL)
0.57 (0.44–0.61)
0.51 (0.45–0.61)
0.49 (0.43–0.59)
0.55 (0.42–0.64)
Salivary cortisol before dinner (μg/dL)
0.15 (0.09–0.18)* ††
0.11 (0.09–0.25)* ††
0.08 (0.06–0.11)
0.05 (0.05–0.07)
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camp (p = 0.021). The salivary cortisol concentrations upon waking were significantly lower on both days at the 
higher-altitude camp than on day 2 at the lower-altitude camp (Higher-day 1 vs. Lower-day 2, p = 0.038; Higher-
day 2 vs. Lower-day 2, p = 0.025). The concentrations before dinner were also significantly higher on both days 
at the higher-altitude camp than those on both days at the lower-altitude camp (Higher-day 1 vs. Lower-day 1, 
p = 0.026; Higher-day 1 vs. Lower-day 2, p = 0.005; Higher-day 2 vs. Lower-day 1, p = 0.012; Higher-day 2 vs. 
Lower-day 2, p = 0.003).
Rate of change in the salivary cortisol concentrations resulting from exercise in each 
camp. 
Figure 3 presents the comparison of the rate of change in the salivary cortisol concentrations after 
each exercise at both camps. The rates of change in salivary cortisol concentrations were significantly lower dur-
ing the morning exercise than during the afternoon exercise on both days at each camp (Higher-day 1, p = 0.002; 
Higher-day 2, p = 0.002; Lower-day 1, p = 0.005; Lower-day 2, p = 0.008; Fig. 3A,B). After the morning exercise, 
the rate of change in the salivary cortisol concentrations was significantly higher on day 2 at the higher-altitude 
Figure 3.  Comparison of the rate of change in the salivary cortisol concentration resulting from exercise 
between the morning and afternoon time points on days 1 and 2 at the higher-altitude camp (A), lower-altitude 
camp (B), between the morning time points on days 1 and 2 at the higher- and lower-altitude camps (C), and 
between the afternoon time points on days 1 and 2 at the higher- and lower-altitude camps (D). The white box 
plots denote the rates of change in the cortisol concentration at the higher-altitude camp, whereas the gray box 
plots represent those at the lower-altitude camp. The significant differences between two time points of exercise 
for runners were analyzed using the Wilcoxon signed-rank test. 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.
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camp than on day 2 at the lower-altitude camp (p = 0.028; Fig. 3C). After the afternoon exercise, the rate of 
change in the salivary cortisol concentrations was significantly higher on both days at the higher-altitude camp 
than on both days at the lower-altitude camp (Higher-day 1 vs. Lower-day 1, p = 0.012; Higher-day 2 vs. Lower-
day 2, p = 0.003; Fig. 3D).
Discussion
In this study, we demonstrated whether the stress responses of runners in training camps at different altitudes 
could be evaluated via sequential saliva collection and automated salivary cortisol measurement. These methods 
were able to detect the basal levels and exercise-induced changes in the salivary cortisol within the runners’ cir-
cadian rhythms at each altitude camp. The basal salivary cortisol concentrations before dinner were significantly 
higher at the higher-altitude camp than at lower-altitude camp. The rate of change in the salivary cortisol concen-
trations during the afternoon exercise on days 1 and 2 and the indicators of exercise intensity were significantly 
higher at the higher-altitude camp than at the lower-altitude camp. Moreover, the rate of change in the salivary 
cortisol concentrations during the morning exercise on day 2 was significantly higher at the higher-altitude camp 
than at lower-altitude camp; no differences were observed in the exercise programs and intensities, such as the 
running distances, velocities, and Borg scale scores.
There have been contradictory reports with regard to the effects of altitude training on cortisol secretions. In 
male elite climbers, the resting serum cortisol and plasma adrenocorticotropic hormone (ACTH) levels taken 
at 07:00–07:30 did not change at 5200 m extreme altitude camp compared with those at sea  level6. In male and 
female elite skiers, no significant differences were observed in resting salivary cortisol concentrations taken 
at 07:00–08:00 between a control group training and living at an altitude of 1200 m and another group train-
ing at 1200 m but living at a simulated altitudes of 2500 m, 3000 m, and 3500 m for 6 days in hypoxic  rooms7. 
In contrast, the basal concentrations of serum cortisol in the morning after 3–4 days at 4350 m were elevated 
compared with those at sea level in healthy men, but this was not statistically  significant8. In the present study, a 
significant difference was observed in the basal salivary cortisol concentrations before dinner between the two 
camps with an altitude difference of approximately 300 m, even at the relatively low altitudes of 1300–1800 m. 
These differences may be due to the fact that the lowest cortisol levels were compared in the evening, whereas 
the previous studies compared the concentrations in the  morning6–8. Another reason may be that the athletes 
were acclimatized to these altitudes when transitioning from the first higher-altitude camp to the second lower-
altitude camp. Overtraining syndrome causes a reduced cortisol response to exercise and changes in the circa-
dian rhythms of cortisol, including low resting levels and peak loss after  waking21,22. In the present study, the 
runners’ salivary cortisol concentrations were lower in the evening, but peaked after waking and increased after 
high-intensity exercise. The low levels of salivary cortisol in the evenings indicated that the runners were not 
suffering from overtraining syndrome but rather were able to adapt to the higher altitudes. Although further 
analysis under higher-altitude conditions is required, it may be more useful to evaluate the cortisol levels in the 
evening rather than in the morning using serum or saliva for assessing the acclimatization to the several altitude 
environments among athletes.
Regarding the acute response to exercise, the increase in cortisol levels after interval training at 09:00 exhibited 
an insignificant trend toward higher values at an altitude of 1800 m when compared with that observed at near 
sea levels in highly trained endurance  athletes9. Another study found that the serum cortisol levels significantly 
increased after resistance training at 70% of the maximum strength under 13% hypoxic conditions from 08:00 
to 11:30 but not under normoxic conditions in healthy male  subjects10. Conversely, the serum cortisol levels did 
not change after resistance training at 50% of the maximum strength under hypoxic and normoxic  conditions11. 
In the present study, the rates of change in the salivary cortisol concentration after the morning exercise were 
significantly lower than those after the afternoon exercise on both days at each altitude camp due to the influence 
of the circadian rhythm, which was validated by previous  reports12,16. In contrast , the comparison of the rates of 
change in cortisol concentration during exercise at the same time on different days, whether in the morning or 
in the afternoon, was effective in evaluating the stress responses, as previously  described16. In the present study, 
we were able to detect the difference in the rates of change in the salivary cortisol concentration during exercise 
with the same program at different altitudes, even in the morning. Moreover, we could detect the increase in 
stress due to the differences in altitude and exercise intensity, more clearly in the afternoon, by assessing the 
rate of change in salivary cortisol concentrations. It was revealed that an increase in the training altitude of 
approximately 400 m at the low altitude of 1300–1700 m with high-intensity exercise resulted in an increase in 
cortisol secretion in both morning and afternoon. This result validates the previous studies, where the serum 
cortisol concentrations were acutely elevated by high-intensity exercise with higher  VO2 max
4,5. However, we did 
not measure the oxygen saturation  (SpO2) or  VO2 max as the oxygen tolerability of runners for different exercise 
intensities. Future studies should evaluate the relationships between the changes in salivary cortisol concentra-
tions and these oxygen tolerability markers in response to endurance exercises at different altitudes. Addition-
ally, the range of the runners’ salivary cortisol concentrations was broad, especially after afternoon exercise on 
day 2 at the higher-altitude camp, shown in Fig. 2. This broad range suggested individual differences in stress 
responses induced by the altitude training in the runners. Therefore, the cortisol concentration measurement 
technique described in this study which uses continuous saliva collection during the circadian rhythms would 
be more useful as a personalized conditioning tool to detect the differences in stress responses under various 
environments for an individual athlete, rather than as a statistical analysis tool for a large number of athletes.
This study has several limitations. First, the sample size was relatively small. We focused on enrolling well-
trained female runners with standardized meal and sleep times during the two consecutive camps. Second, the 
sequential saliva collections and measurements of salivary cortisol concentration were not performed at sea level. 
The temperatures were around 20 °C during both altitude camps, but the temperature during the same period 
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was as high as 30–40 °C at near sea level where the runners lived and trained. Previous research found that the 
salivary cortisol levels detected in a maximal progressive test using a cycle ergometer were significantly higher 
under the hot conditions (40 °C) than under normal conditions (22 °C) in nine young healthy  men23. Therefore, it 
was impossible in this study to collect saliva at near sea level without the stress resulting from high temperatures. 
Future study should utilize hypoxic rooms, in which the environmental conditions, including temperature, are 
standardized. Additionally, more runners should be enrolled, specifically males.
Conclusions
Measurement of the salivary cortisol levels within the circadian rhythm led to the detection of the changes in the 
stress response due to the same intensity exercise at different altitudes, even in the morning. Additionally, evening 
resting salivary cortisol levels can be used to assess athletes’ acclimatization to high altitudes. The combination 
of sequential saliva collection and automated cortisol measurements may be useful for assessing adaptation dis-
orders and excessive exercise stress, and also may help develop adequate altitude training programs for athletes.
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on 
reasonable request.
Received: 3 February 2022; Accepted: 31 May 2022
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Acknowledgements
We are grateful to Kenichi Morikawa, Mai Murata, Mayumi Nishiyama, and Tomoyuki Aoki for providing tech-
nical assistance and helpful discussions.
Author contributions
K.T. participated in the collection and analysis of data and manuscript writing, reviewing and editing. K.U., L.M., 
A.N., R.H., R.S., N.S., A.Y., N.K., and T.K. participated in data collection and analysis. M.M. participated in con-
ception of the study, supervision, and manuscript editing. All authors read and approved the final manuscript.
Funding
This work was supported by the Ministry of Education, Culture, Sports, Science, and Technology of Japan [Grant 
number 18K07406].
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Competing interests 
The authors declare no competing interests.
Additional information
Correspondence and requests for materials should be addressed to K.T.
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