Objective: The present research aimed to analyse the autonomic, anxiety, perceived exertion, and self-confidence response during real and simulated flights. Methods: This cross-sectional study participated 12 experienced male pilots (age = 33.08 (5.21)) from the Spanish Air Force. Participants had to complete a real and a simulated flight mission randomly. The heart rate variability (HRV), anxiety, self-confidence, and rating of perceived exertion were collected before and after both manoeuvres, and HRV was also collected during both simulated and real flights. Results: When studying the acute effects of real and simulated flights, the mean heart rate, the R-to-R interval, the cognitive anxiety and the perceived exertion were significantly impacted only by real flights. Furthermore, significant differences in the mean heart rate and RR interval were found when compared to the acute effects of real and simulated flights (with higher acute effects observed in real flights). Additionally, when compared the HRV values during simulated and real flights, significant differences were observed in the RR and heart rate mean (with lower RR interval and higher heart rate mean observed during real flights). Conclusion: Real flights significantly reduced the RR interval and cognitive anxiety while increased the heart rate mean and the rating of perceived exertion, whereas simulated flights did not induce any significant change in the autonomic modulation.