PURPOSE: The occupation of the emergency physicians (EPs) of helicopter emergency medical services (HEMS) can be characterized as a high-strain occupation (Karasek in Adm Sci Q 24(2):285-308. https://doi.org/10.2307/2392498 , 1979). Therefore, the aim of this study was to measure and compare the stress load of the EPs of HEMS on duty on air ambulance workdays and on 2 control days. METHODS: In this field study (within-subjects design), hormonal, physiological, and self-perceived stress levels of 20 EPs [3 females, 17 males; mean age (M) = 44.95 years, SD = 4.80, 95% confidence interval (CI) (42.71, 47.19)] of HEMS, were recorded on different test days. Measurements of the cortisol awakening response (CAR) and the heart rate variability (HRV) were performed while on duty on the air ambulance and during workdays at the outpatient clinic as well as at home on days of rest. RESULTS: There were significant differences in the CAR (area under the curve with respect to ground F(2,38) = 12.81, p < 0.001) between the 3 test days with the highest values on the workday at the outpatient clinic [M = 81.24; 98.75% CI (61.24, 101.24)] and not on the air ambulance day [M = 61.82; 98.75% CI (45.18, 78.46)] or on the day of rest [M = 52.96; 98.75% CI (38.17, 67.76)]. In addition, the HRV parameter SDNN [F(2,38) = 6.369; p = 0.004] presented significant differences between the 3 test days with lower levels on the day at the outpatient clinic [M = 101.44; 98.75% CI (83.50, 119.38)] in contrast to the air ambulance day [M = 120.16; 98.75% CI (100.02, 140.30)] and to the resting day [M = 123.79; 98.75% CI (106.49, 141.10)]. Furthermore, there were significant differences in the HRV parameter LF/HF [F(2,38) = 6.215; p = 0.005] between the 3 testing days with the highest values on the workday at the outpatient clinic [M = 8.69; 98.75% CI (6.29, 11.09)] compared to the air ambulance day [M = 6.54; 98.75% CI (4.50, 8.57)] and the day of rest [M = 6.43; 98.75% CI (4.57, 8.29)]. CONCLUSIONS: Compared with the standard values and previous studies, EPs of HEMS have an increase in hormonal reactivity in the morning and a lack of recovery of the ANS. It can be concluded that-with respect to the psychobiological stress model by McEwen and Lasley (The end of stress as we know it, National Academic Press, Washington, 2003)-work-related stressors persist too long or the stress response is exaggerated (allostatic load) due to chronic stress induction and lack of recovery.