Objectives: The aim of this article is to investigate the psychophysiological responses of unmanned aerial vehicle (UAV) pilots during flight simulations with high mission complexity. In particular, it focuses on the responses of the sympathetic and parasympathetic parts of the autonomic nervous system. Material and Methods: Fourteen pilots aged 26–31 years took part in the study. The research was conducted using a UAV flight simulator. During the test, data was collected from electrocardiogram sensor and piezoelectric (lead zirconate titanate – PZT) respiration sensor as well as the pilot’s mission performance was assessed. In addition, the test subjects were subjected to a simple reaction speed test after the completed exercise. Results: For missions classified as having low difficulty, the mean RR interval (meanRR) was 1004.03 milliseconds, with a standard deviation (SD) = 18.5 ms. This corresponds to an mean heart rate of about 59.8 bpm (SD = 1.1), which is 16.9% longer than the RR intervals observed during high difficulty missions (meanRR±SD 859±59.75 ms). The values of respiratory rate per minute for the different levels of difficulty were M±SD 17.3±0.87 for low, 18.1±1.04 for medium, and 18.8±0.41 for high mission difficulty. Conclusions: A correlation between the effects of an unmanned aircraft flight simulator and the pilot’s body was proven. By means of tests using electrocardiogram, PZT and reaction time measurements, it was proven that the flight simulator directly induces stressful stimuli that affect the subject’s body. By analyzing the individual results, it was also proven that the sympathetic part of the nervous system is activated as the level of mission difficulty increases. Int J Occup Med Environ Health. 2025;38(4):391–404