A Prospective Cohort Study on the Effects of Repeated Acute Stress on Cortisol Awakening Response and Immune Function in Military Medical Students

Abstract

Background: The cortisol awakening response (CAR) is a pivotal component of the body's stress response, yet its dynamics under repeated acute stress and its interplay with immune biomarkers remain inadequately understood. Methods: This study examined 80 second-year military medical students undergoing a 5-day intensive surgical simulation designed to elicit stress responses. Salivary samples were collected daily upon waking and 30 min thereafter to measure cortisol and a panel of cytokines using bead-based multiplex ELISA. Results: Analysis revealed a significant blunting of the CAR on the third day of training (p = 0.00006), followed by a recovery on the fourth day (p = 0.0005). Concurrently, specific cytokines such as CXCL1 (r = 0.2, p = 0.0005), IL-6 (r = 0.13, p = 0.02), IL-10 (r = 0.14, p = 0.02), and VEGF-A (r = 0.17, p = 0.003) displayed patterns correlating with the CAR, with increased strength of associations observed when assessing cytokine levels against the CAR of the preceding day (CXCL1 r = 0.41, p = 0.0002. IL-6 r = 0.38, p = 0.0006. IL-10 r = 0.3, p = 0.008. VEGF-A r = 0.41, p = 0.0002). Conclusions: These results suggest a temporal relationship between stress-induced cortisol dynamics and immune regulation. The CAR pattern demonstrated in this study may represent induction of and recovery from psychological burnout. Moreover, the observed cytokine associations provide insight into the mechanisms by which stress can influence immune function. The results may have broader implications for managing stress in high-performance environments, such as military and medical professions, and for identifying individuals at risk of stress-related immune suppression.

Keywords: cortisol awakening response (CAR); hyper-realistic surgical simulation; immune function; repeated acute stress; salivary cytokines; stress physiology.

Figure 1

Intensive Surgical Skills Week schedule. Samples were collected at wake and 30 min post waking. Students go through hyper-realistic mass casualty and operating room simulations, going through each simulation two times over the duration of training.

Figure 2

Daily mean percent change in AM salivary cortisol from waking to 30 min later (CAR) across all participants (n = 79) during repeated acute stress. Error bars represent the standard error of the mean (SEM). Results of pairwise comparisons between daily means are shown by the compact letter display. Two means with the same letter are not significantly different at the 0.05 level. Day 3 CAR is significantly reduced compared to all other days (D1, D3 p = 0.014), (D2, D3 p = 0.00006), (D4, D3 p = 0.0005). Day 1 average CAR = 67.28%, Day 2 = 84.45%, Day 3 = 21.30%, Day 4 = 78.83%. Typically, a healthy CAR is expected to be between 50 and 80%.

Figure 3

Panels A-H represent daily average salivary interleukin levels in pg/mL at Time 1 (waking) and Time 2 (30 min post waking) during repeated acute stress. Error bars represent the SEM. Results of pairwise comparisons between average cytokine values at each time point are shown by the compact letter display. Two means with the same letter are not significantly different at the 0.05 level. All interleukins exhibited significant decreases in average salivary concentration from Time 1 to Time 2 in all days of the sampling period. IL-1a, IL-8 Il-15, IL-18, and IL-1RA, p < 0.0001. IL-6 Day 1, p = 0.0014; Day 2, p = 0.0124; Day 3, p = 0.0003; Day 4, p < 0.0001. IL-10 Day 1, p = 0.003; Day 2, p = 0.0056; Day 3, p = 0.0086; Day 4, p = 0.0098. IL-1B Day 1, p = 0.0001; Days 2–4, p < 0.0001. (a) IL-1a (n = 79), (b) IL-6 (n = 79), (c) IL-8 (n = 79), (d) IL-10 (n = 79), (e) IL-15 (n = 79), (f) IL-18 (n = 79), (g) IL-1β (n = 40), (h) IL-1RA (n = 79).

Figure 4

Panels A-E represent daily average salivary growth factor levels in pg/mL at Time 1 (waking) and Time 2 (30 min post waking) during repeated acute stress. Error bars represent SEM. Results of pairwise comparisons between average cytokine values at each time point are shown by the compact letter display. Two means with the same letter are not significantly different at the 0.05 level. VEGF-A showed significant increases in the post wake period on all 4 days (p = 0.0059, p = 0.0105, p < 0.0001, p = 0.0155). PDGF-AA showed significant increases in the post wake period on Days 3 and 4 (p = 0.0001, p = 0.0005). EGF, showed significant decreases in the post wake period on all days (p < 0.0001). FGF-2 showed significant decreases on Days 1–3 (p < 0.0001, p = 0.0003, p = 0.008). TGFα showed a significant decrease on Day 3 (p = 0.011). (a) EGF (n = 79), (b) FGF-2 (n = 79), (c) VEGF-A (n = 79), (d) TGFα (n = 79), (e) PDGF-AA (n = 79).

Figure 5

Panels A-F represent daily average salivary chemokine and miscellaneous cytokine levels in pg/mL at Time 1 (waking) and Time 2 (30 min post waking) during repeated acute stress. Error bars represent SEM. Results of pairwise comparisons between average cytokine values at each time point are shown by the compact letter display. Two means with the same letter are not significantly different at the 0.05 level. MCP-1 showed significant decreases in the post wake period on all 4 days (p < 0.0001). CX3CL1 showed decreases in salivary concentration in the post wake period on all days but only the decrease on Day 2 reached statistical significance (p = 0.0198). TNFα showed significant decreases in salivary concentration from Time 1 to Time 2 on all days (p = 0.0021, p = 0.0055, p < 0.0001, p = 0.0028). G-CSF levels do not show significant changes from Time 1 to Time 2 on any day; however, overall levels are significantly decreased between Day 3 and Day 4 (T1 p = 0.0028, T2 p = 0.0001). (a) CXCL1 (n = 79), (b) CX1CL3 (n = 40), (c) MCP-1 (n = 79), (d) IFNγ (n = 40), (e) TNFα (n = 40), (f) G-CSF (n = 40).