In this study, we investigated whether the automated measurement of the salivary testosterone and cortisol concentrations and the salivary T/C ratio can be used to assess the stress induced by exercise at different intensities among male long-distance runners accurately and effectively while considering circadian rhythms. The salivary testosterone and cortisol concentrations showed positive correlations with their respective serum concentrations. The combination of sequential saliva collection and automated ECLIA measurement was able to detect the circadian rhythms of the testosterone and cortisol concentrations and the T/C ratio, as well as acute changes caused by exercise. However, measurable saliva samples could not be obtained via passive drooling from five participants due to low volume and high viscosity. The IT group showed a significantly higher rate of change in the salivary cortisol concentration and a significantly lower rate of change in the salivary T/C ratio during interval training in the evening on day 1, as well as the multiple intensity indices. Such changes were not observed in the salivary testosterone concentration.
The cotton swab is a convenient method for quickly collecting a sufficient volume of saliva without residue and mucus for assessing stress marker levels14. The cortisol concentration in cotton swab samples is a better predictor of the serum total cortisol and free cortisol concentrations than passive drooling among healthy volunteers7. In contrast, cotton swab samples have falsely high testosterone concentrations when assessed by immunoassays15,16. Previous studies have shown that the cross-reactivity between plant hormones and antibodies influence the results of the testosterone immunoassay if a cotton swab is used15,16,17,18. This is why we used passive drooling in the current study. The salivary cortisol concentration measured by automated second-generation ECLIA has a significantly positive correlation with the measurement by liquid chromatography–tandem mass spectrometry (LC–MS/MS)19. We previously assessed the exercise-induced stress among female long-distance runners by combining automated measurement of the salivary cortisol concentration with sequential sampling using a cotton swab9,10. In that study, the salivary cortisol concentration from ECLIA showed a significantly positive correlation with the concentration from an enzyme-linked immunosorbent assay9. Moreover, all salivary samples collected by a cotton swab could be measured without some needing to be excluded due to a low dose or high viscosity samples9,10.
In the present study, we first evaluated the salivary testosterone concentration using ECLIA by comparing them to the serum samples. The salivary testosterone concentration showed a significantly more positive correlation with the serum free testosterone concentration than with the serum total testosterone concentration. The salivary testosterone concentration can be combined with sequential sampling using passive drooling to assess the circadian rhythm among runners. However, the saliva samples of several participants that were collected via passive drooling did not have sufficient volume and were extremely viscous, so they could not be utilized for ECLIA. Physical and mental stresses reduce the flow rate and increase the viscosity of saliva20,21. Assays including ECLIA require a sufficient sample volume (minimum of 100–200 µL) because of the dead volume required for trouble-free automated sample processing. Automated ECLIA for measuring the salivary cortisol and testosterone concentrations is advantageous because it can measure a large number of samples easily and rapidly. However, its usefulness may be reduced if used in combination with passive drooling owing to its requirement for large sample volumes without residue and mucus.
Previous studies have shown that the serum cortisol concentration acutely increases due to moderate- to high-intensity endurance exercise (i.e., > 60% of the maximal oxygen consumption [VO2 max])22,23. Sato et al.24 showed that the serum cortisol and free testosterone concentrations in healthy young men were elevated by two 15-min sessions of submaximal exercise using an electromechanically braked ergometer at ≥ 40% of their peak oxygen uptake (VO2 peak) among non-athletes. However, the serum cortisol and free testosterone concentrations were only increased among male endurance runners by exercise at 90% VO2 peak. Tremblay et al.25 showed that a running duration of at least 80 min increases testosterone and cortisol concentrations during low-intensity endurance exercise. Resistance exercise acutely increases testosterone secretion, which is an anabolic hormone that is essential for muscular adaptation and muscle growth1. In a previous meta-analysis, Hayes et al.5 revealed that although acute aerobic and resistance exercises consistently increase the salivary testosterone concentration, the acute response of salivary testosterone to power-based exercise has not been fully elucidated. Anderson et al.26 showed that the serum free testosterone concentration decreases immediately after exhaustive endurance exercise and gradually increases after 24 h or during the recovery process among male endurance athletes.
In the current study, the interval training during the evening on day 1 for the IT group had the highest exercise intensity based on indicators including the running velocity, Borg scale score, and maximum pulse rate. The interval training significantly increased the salivary cortisol concentration and decreased the salivary T/C ratio. The rate of change in the salivary testosterone concentration showed no significant differences for a given exercise between the two groups or between different exercises within the same group. The salivary testosterone concentration increased after the evening exercise on both days for both groups, despite the differences in exercise intensity. The circadian rhythm of serum testosterone is characterized by high concentrations in the morning followed by a gradual decline in the evening, accompanied by a mild rise from 16:00 to 19:00 pm in young men27. In our study, the increase in salivary testosterone concentrations after evening exercise at 18:30 pm in all runners, independent of exercise intensity, may have detected this circadian rhythm. Moreover, the change in the salivary testosterone concentration may have been affected by a longer exercise duration. Doan et al.28 observed similar results in the circadian rhythm during a 36-hole golf competition, in which the salivary testosterone concentration only increased during holes 25–30 in the evening on the competition day compared with the baseline day. In contrast, the salivary cortisol concentration increased and the salivary T/C ratio decreased at almost every hole on the competition day. They concluded that a low T/C ratio was correlated with good golf performance. The T/C ratio is generally an indicator of the anabolic/catabolic balance during skeletal muscle destruction and recovery29. In the current study, we observed that a lower salivary T/C ratio might reflect the acute stress response to exercises of different intensities. However, we did not assess differences in the performance of participants or changes in hormone levels during recovery. Thus, further study is need on the salivary testosterone concentration and T/C ratio to investigate their associations with the performance and recovery processes of endurance- and resistance-trained athletes.
Detecting the responses of testosterone and cortisol to exercise in the morning is a challenge owing to their respective circadian rhythms. However, such measurements are easily obtained in the evening30. We9 previously showed that differences in the rate of change in the salivary cortisol concentration caused by exercise at different intensities could be compared at the same time on different days, even in the early morning. This method allowed us to assess the differences in acclimatization and exercise stress between two altitudes10. In the current study, the rates of change in the salivary testosterone and cortisol concentrations and the T/C ratio caused by morning exercise showed no differences between each group on both days. This may be because the exercise intensity was not sufficient to elicit a hormonal response in this study compared with the exercises used in our previous studies9,10. However, we did observe significant differences in the running velocity and maximum pulse rate in the morning. Further studies involving other types of exercises and higher intensities must be conducted to compare the changes in the hormonal response to exercise in the early morning. In contrast, the rates of change in the cortisol concentration and T/C ratio caused by evening exercise showed significant differences between the two days depending on the exercise intensity. This result suggests that automated salivary cortisol assessment to compare exercise-induced stress response in our previous studies is also useful in determining the salivary T/C ratio. Based on its circadian rhythm, the T/C ratio was lowest after wakeup and then gradually increased. We believe this reflects the circadian rhythm of cortisol rather than that of testosterone. The rate of change in the salivary T/C ratio indicated differences in the stress response between each exercise program. The salivary T/C ratio decreased on day 1 and increased on day 2 after the evening exercise for the IT group. We concluded that it was more influenced by the cortisol response than the testosterone response. In their meta-analysis, Hayes et al.5 obtained similar results showing that the response of the salivary T/C ratio to exercise was due to changes in the salivary cortisol concentration. Because passive drooling may not be sufficient for obtaining the samples required for assessing the T/C ratio, further study should be conducted to evaluate whether cortisol alone can be used to evaluate the exercise-induced stress response. This would be very helpful because cortisol can be measured by using saliva conventionally collected with cotton swabs.
The current study had several limitations. First, the sample size of each group was relatively small. Furthermore, the number of participants in the IT group was reduced because some saliva samples obtained via passive drooling could not be used for the automated measurement. In addition, we were unable to compare the circadian rhythms between sedentary participants without exercise effect and the exercised runners. The reason for this limitation was the focus on standardizing living conditions that prevented the recruitment of large numbers of well-trained runners or non-runners. Moreover, it was not possible to provide a sedentary period because maintaining the condition of the runners was top priority. Second, the exercise programs were not evaluated by using accurate intensity indicators such as VO2 max. The runner’s exercise conditions could not be tightly controlled. In contrast, we formed two groups with or without high-intensity interval training in which multiple intensity indices were significantly higher, and we consider it important to be able to assess the difference in stress response by salivary cortisol concentration and T/C ratio between higher-intensity interval training and lower-intensity running on different days only in IT group. Further studies are needed to set the sedentary group showing the circadian rhythms without exercise effect, and to use a standardized exercise program with more participants and accurate indicators for both the high- and low-intensity exercise. Third, the time between the post-evening exercise and pre-dinner sampling points was not sufficient. Some participants had high salivary hormone concentrations at the pre-dinner sampling point, which should be when they are lowest particularly for cortisol. More studies should be performed to adjust the collection time according to the training program, such as including a point before bedtime to assess the basal concentrations at night.
In conclusion, automated ECLIA assessment of salivary testosterone and cortisol concentrations is as accurate as an assessment using serum samples. The cortisol concentration and T/C ratio assessed via sequential saliva collection and automated evaluations can adequately reflect differences in endurance exercise intensity on different days performed at the same time. Such an approach may be useful for detecting different stress responses among athletes while considering the circadian rhythm.