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Understanding the operator's cognitive workload is crucial for efficiency and safety in human-machine systems. This study investigated how cognitive workload modulates cardiac autonomic regulation during a standardized military simulator flight. Military student pilots completed simulated flight tasks in a Hawk flight simulator. Continuous electrocardiography was recorded to analyze time and frequency domain heart rate variability (HRV). After the simulation, a flight instructor used a standardized method to evaluate student pilot's individual cognitive workload from video-recorded flight simulator data. Results indicated that HRV was able to differentiate flight phases that induced varying levels of cognitive workload; an increasing level of cognitive workload caused significant decreases in many HRV variables, mainly reflecting parasympathetic deactivation of cardiac autonomic regulation. In conclusion, autonomic physiological responses can be used to examine reactions to increased cognitive workload during simulated military flights. HRV could be beneficial in assessing individual responses to cognitive workload and pilot performance during simulator training., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)