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OBJECTIVE: We evaluated a variety of non-invasive physiological technologies and a series of test approaches for examination of aviator performances under conditions of mental workload in order to provide a standard real-time test for physiological and psychological pilot fatigue assessments. METHODS: Twenty-one male aviators were selected for a simulated flight in a hypobaric cabin with artificial altitude conditions of 2400 meter above sea level. The simulated flight lasted for 1.5 h, and was repeated for two times with an intervening 0.5 h rest period outside the hypobaric cabin. Subjective criteria (a fatigue assessment instrument [FAI]) and objective criteria (a standing-position balance test as well as a critical flicker fusion frequency (CFF) test) were used for fatigue evaluations. RESULTS: No significant change was observed in the FAI scores before and after the simulated flight, indicating that there was no subjective fatigue feeling among the participants. However, significant differences were observed in the standing-position balance and CFF tests among the subjects, suggesting that psychophysiological indexes can reflect mental changes caused by workload to a certain extent. The CFF test was the simplest and clearly indicated the occurrence of workload influences on pilot performances after a simulated flight. CONCLUSIONS: Results showed that the CFF test was the easiest way to detect workload caused mental changes after a simulated flight in a hypobaric cabin and reflected the psychophysiological state of aviators. We suggest that this test might be used as an effective routine method for evaluating the workload influences on mental conditions of aviators.