Helicopter emergency medical service (HEMS) is an essential part of prehospital emergency medicine. The working conditions lead to high physical stress, especially in rescue operations. The study aimed to determine the cardiovascular stress profile during rescue situations in HEMS crew members. Twenty-one HEMS crew members (male n = 20) participated in the prospective study. Heart rate, blood pressure and long-term ECG measurements were recorded during the whole operation day. The changes of measurements during rescue operation (52 operations in total) were compared to these of standby time. Rescue operations lead to increased load on the cardiovascular system, as expressed by significantly higher blood pressure, heart rate values and rate of cardiac events compared to standby time. Of special note, the difference in systolic blood pressure mean was 7.4 ± 9.0 mmHg (CI [5.1; 9.7], p < 0.001). Maximal heart rate was on average 33.7 bpm higher during rescue operation than in the standby time (CI [26.2; 40.8], p < 0.001). Cardiac events occurred significantly more frequently during the period of rescue operation than in standby time hours (p = 0.02). The results reported a significant load on the cardiovascular system during rescue operations in HEMS crew members. Therefore, it is necessary to carry out a risk stratification of the HEMS crew members to prevent cardiovascular risk and events.