Objective: Exercise with virtual reality (VR) is a novel approach to the promotion of health and the components of fitness. Our objective was to investigate the effects of exercise with VR on physical performance, mental fatigue, and physical fatigue in single-stage aerobic and anaerobic running.Materials and Methods: Twenty physically active, healthy, age-matched male university students executed the exercise test in aerobic and anaerobic groups in two separate sessions of VR (first-person perspective in a simulator) and non-VR (traditionally in the laboratory). The simulated environment was a graphic tunnel route. The aerobic and anaerobic groups engaged in the Bruce protocol and anaerobic speed test, respectively. Exercise test duration, blood serum lactate concentration, rating of fatigue (ROF), and heart rate were recorded at the end of both sessions.Results: Intragroup comparisons using the dependent t-test indicated that the exercise test duration significantly increased in the VR session of the anaerobic group compared to the traditional method considering the effect size (t7=0.804; P=0.804). However, the test duration remained unchanged in both sessions of the aerobic group (t9=1.373; P=0.203). Serum lactate concentration significantly decreased in the VR session compared to the non-VR session in the aerobic group (t9=4.734; P=0.001) and anaerobic group (t9=2.321; P=0.045). ROF and heart rate of the aerobic and anaerobic groups remained unchanged in both sessions.Conclusion: The findings indicated that as an alternative for improving physical performance in exercise, running with VR in an imaginative virtual environment decreased physical fatigue in a single stage of aerobic and anaerobic running.