Presented work is primarily oriented on the experimental verification of the influence of fatigue on the psychological condition of the flying personnel, using psychological and performance tests. For the evaluation of a pilot performance, the 24 hours experiment was conducted. In total, eight subjects participated in the experimental measurements. Eight participants went through several tests, including simulator flights, to investigate the effects of the fatigue on the results of psychological measurements. Measurements included workload evaluation, using NASA task load evaluation concept and performance testing, using the so-called OR-test. Significant statistical differences between measurements performed during 24 hours were not found in the case of NASA task load Scores. In the case of OR-test, Friedman ANOVA and subsequent post-hoc analysis show that the greatest decrease in performance was observed in approximately 22 hours of wakefulness, i.e. approximately in half of the measuring process. The concept of 24-hour measurements for the quantification of fatigue is not commonly used yet as well as objectivization using performance testing. As the apparent effect of fatigue is mainly on performance testing results, it can be argued that this work could serve as a basis for further studies on fatigue. Also, it could serve as a support for introducing new pilots' psychological testing procedures in the future, which could contribute to current efforts to improve aviation safety.