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 high in the second phase as well, but decreased to baseline level in the third and fourth phases, even though these phases were designed to be the most stressful. Cadets classified as threat responders based on physiological data (n = 3) scored significantly higher on subjective threat/challenge appraisal than those classified as challenge responders (n = 21). It seems that in the tested VR training scenario, the novelty of the scenario was the only effective stress stimuli, whereas the other implemented stressors did not cause a measurable physiological response. We conclude that if VR training scenarios are to be used for stress training, these should confront trainees with unpredictable but context-specific demands.@en