Virtual reality (VR) provides the ability to simulate stressors to replicated real-world situations. It allows for the creation and validation of training, therapy, and stress countermeasures in a safe and controlled setting. However, there is still much unknown about the cognitive appraisal of stressors and underlying elements. More research is needed on the creation of stressors and to verify that stress levels can be effectively manipulated by the virtual environment. The objective of this study was to investigate and validate different VR stressor levels from existing emergency spaceflight procedures. Experts in spaceflight procedures and the human stress response helped design a VR spaceflight environment and emergency fire task procedure. A within-subject experiment evaluated three stressor levels. Forty healthy participants each completed three trials (low, medium, high stressor levels) in VR to locate and extinguish a fire on the International Space Station (VR-ISS). Since stress is a complex construct, physiological data (heart rate, heart rate variability, blood pressure, electrodermal activity) and self-assessment (workload, stress, anxiety) were collected for each stressor level. The results suggest that the environmental-based stressors can induce significantly different, distinguishable levels of stress in individuals.