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With developing efforts to increase aviation safety, the focus on human factors is being emphasized as well. One of the phenomena affecting the performance of the pilot or other aviation specialists is undoubtedly the fatigue. Dangerous situations can occur, especially when fatigue accumulates. Monitoring fatigue is therefore in the interest of all stakeholders as well as the aviation authorities, which have developed Fatigue Risk Management System. In spite of the fact that the system itself at its highest level also uses the objective data, to this day, the monitoring of fatigue in aviation has mainly been based on a questionnaire survey. Although, the standardized NASA Task Load Index (NASA TLX) is often applied, it remains questionable whether such a questionnaire realistically reflects the fatigue. Based on previously mentioned, the NASA TLX was modified to focus on pilot fatigue and then applied during 24-hour measurements for the purposes of this paper. Eight pilots took part in the measurement, each of them performed 8 simulated flights. From the results of the questionnaire survey, a window of circadian low can be observed, in accordance with the evaluated theta/alpha parameter obtained by spectral analysis of EEG records. The results therefore indicate that such a questionnaire is able to indicate fatigue.