Changes in the cortisol concentration
Data from 45 of the 46 participants were included in the analysis of measured cortisol concentrations (15 m, 14 w, 16 woc). There was a 1.6-fold increase in cortisol levels after the psychosocial stressor (T2) and a 1.9-fold increase in cortisol levels after the physical stressor (T2) (Supplementary Fig. 1). The ANOVA revealed a main effect of time of measurement (F(1.68, 70.64) = 23.75, p < .001, η2p = .36) and an interaction between time of measurement and stressor (F(1.69, 71.04) = 5.19, p = .01, η2p = .11). None of the other effects were significant. Subsequent post-hoc tests for the two-way interaction effect within stress types revealed significant differences between time points for the psychosocial (F(1.56, 68.5) = 16.6, p < .001, η2p = .27) and physical stress tests (F(1.69, 74.5) = 16.2, p < .001, η2p = .27).
Changes in α-amylase levels
For the analysis of α-amylase levels, data from all 46 participants were included (15 m, 15 w, 16 woc). There was a 1.2-fold increase in α-amylase after the physical stressor (T2). For the psychosocial stressor, α-amylase levels decreased by a factor of 1.2 from T1 to T3 (Supplementary Fig. 2). Analysis revealed a significant main effect of time point (F(3, 129) = 16.27, p < .001, η2p = .28) and a significant interaction effect of stressor by time point (F(3, 129) = 6.21, p < .001, η2p = .13). In addition, post-hoc analysis revealed a significant main effect for differences between time points in the psychosocial (F(2,5, 112) = 3.07, p = .04, η2p = .06) and physical stress condition (F(3, 135) = 16.6, p < .001, η2p = .27).
Changes in the cfDNA concentration in plasma
Plasma cf-nDNA
The analysis of cf-nDNA includes data from 44 of the 46 participants (15 m, 13 w, 16 woc). Descriptive analysis of the data showed a peak in cf-nDNA concentration at T2 for the physical stressor. Cf-nDNA increased by a factor of 2.2 and then decreased steadily. For the psychosocial stress paradigm, cf-nDNA levels remained nearly unchanged (Supplementary Fig. 3A).
Cf-nDNA levels increased significantly after both stressors (main effect time: F(2,47,101,39) = 27.44, p < .001, η2p = .4). There were significant differences in cf-nDNA levels (magnitude and response dynamics) over time between the two stressors (main effect stressor type: F(1, 41) = 5.59, p = .02, η2p = .12; time x stressor type: F(3,123) = 7.86, p < .001, η2p = .16). Additional analyses using post hoc tests showed that these differences occurred for both the psychosocial and the physical stressor (interaction time x stressor-psychosocial stressor: F(3, 129) = 6, p < .001, η2p = .12; physical stressor: F(2,57, 111) = 28.2, p < .001, η2p = .4). Compared to psychosocial stress, physical stress led to an overall greater increase in cf-nDNA over time. Furthermore, there were significant differences in cfDNA levels between groups over time for both stress conditions (group x stressor: F(2, 41) = 3.49, p = 0.04, η2p = 0.15; time x group x stressor: F(6, 123) = 2.31, p = .04, η2p = .1). The main effect of group and the interaction between group and time were not significant. Group specific ANOVAs were performed for further analysis. All three groups showed significant changes in cf-nDNA over time (main effect time group m: F(3, 42) = 13.2, p < 0.001, η2p = 0.49; group w: F(1.55, 18.6) = 4.89, p = 0.03, η2p = 0.29; group woc: F(3, 45) = 12.8, p < .001, η2p = .46). In addition, the cf-nDNA response of male participants differed significantly between the two stressors (main effect stressor group m: F(1, 14) = 10.2, p = .006, η2p = .42). Moreover the response of the cf-nDNA to both stressors was different in the male group as well as in the female group with oral hormonal contraception over time (interaction time x stressor group m: F(3, 42) = 7.18, p < 0.001, η2p = 0.34; group woc: F(3, 45) = 3.32, p = .03, η2p = .18).
Plasma cf-mtDNA
The analysis of plasma cf-mtDNA included data from 42 of the 46 participants (14 m, 13 w, 15 woc). The descriptive analysis of the data showed a peak in cf-mtDNA at T1 for the physical stressor. The cf-mtDNA decreased by a factor of 2 from T1 to T2 for the physical stressor (Supplementary Fig. 3B). This was followed by a decrease in cf-mtDNA (T2 and T3) and a slight increase in cf-mtDNA at T4. For the psychosocial stressor, cf-mtDNA increased over time with a peak at T4. Throughout the test session, within the psychosocial stress paradigm, cf-mtDNA levels increased by a factor of 1.6 (Fig. 2B). Repeated measures ANOVA revealed a significant main effect of time of measurement (F(3, 117) = 3.6, p = .02, η2p = .09) and an interaction between time of measurement and stressor (F(3, 117) = 4.34, p = .006, η2p = .1). Subsequent post-hoc tests revealed significant differences in plasma cf-mtDNA after physical stress (F(3, 123) = 5.79, p < .001, η2p = .12), but not after psychosocial stress.
Changes in the cfDNA concentration in saliva
Saliva cf-nDNA
Salivary cf-nDNA analysis included data from 42 of the 46 participants (13 m, 15 w, 14 woc). Descriptive analysis revealed a peak in cf-nDNA concentration at T3 for the psychosocial stressor. During the physical stress condition, the cf-nDNA concentration remained similar (Supplementary Fig. 4A). However, for the psychosocial stressor, cf-nDNA increased by a factor of 1.3 (Supplementary Fig. 4A). The repeated measures ANOVA revealed a significant main effect only for the time of measurement (F(2,29,89,36) = 4,28, p = .01, η2p = .1).
Saliva cf-mtDNA
Analysis of salivary cf-mtDNA included the data from 43 of the 46 participants (13 m, 15 w, 15 woc). Descriptive analysis showed similar levels of cf-mtDNA after physical stress and a 1,4-fold increase at T3 after psychosocial stress (Supplementary Fig. 4B). However, no statistically significant effects were observed.
Correlations between cf-nDNA, cf-mtDNA, stress hormone levels and heartrate
In addition to the repeated measures ANOVAs, pre-post differences in all cfDNA markers were calculated and correlated with pre-post differences in cortisol and alpha-amylase levels. In addition, maximum heart rate was correlated with increases in cfDNA markers in the exercise condition. The analysis used data from 36 participants (12 m, 11 w, 13 woc). None of the correlations reached significance.