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The current study explores whether different stressors in a virtual reality (VR) military training scenario cause increases in physiological stress. This would validate the use of VR simulation for stress training, as well as the physiological monitoring of trainees for educational purposes. Military cadets (n = 63) performed a patrol scenario (military convoy) in which they answered questions about their surroundings. Stressors (task difficulty, noise, lighting changes, social evaluations, electric muscle stimulation, and a simulated attack on the convoy) were stepwise added in four phases. Electrocardiogram, blood pressure, electrodermal activity, cortisol, and the cadets’ subjective threat/challenge appraisal were measured. We found that only the first phase caused a significant increase in physiological stress, as measured with heart rate, heart rate variability, and electrodermal activity. Physiological stress appeared to stay …