© by the Aerospace Medical Association, Alexandria, VA. INTRODUCTION: Sleep loss can result in cognitive, motor, and neurobehavioral impairments. In an aviation context, this can cause a serious threat to flight safety. Therefore, the study aimed to investigate the effects of 24-h sleep deprivation on mood, fatigue, and airline pilot competencies. METHODS: Seven subjects attended two 24-h testing periods, one with an 8-h sleep opportunity, and the other with no sleep opportunity (i.e., sleep deprivation). Subjects were required to complete a battery of mood, fatigue, and analogue measures of pilot competency tasks every 8 h (0 h, 8 h, 16 h, 24 h) throughout each testing period. RESULTS: While total mood disturbance was found to significantly increase (83.42, SD = 25.7), both objective (352.71, SD = 42.00) and subjective (34.85, SD = 8.82) fatigue were found to significantly decrease following 24-h sleep deprivation. Cognitive flexibility (757.45, SD = 58.48) and hand-eye coordination (dominant hand only) (60.28, SD = 3.86) were also negatively impacted following 24-h sleep deprivation. However, working memory and situation awareness were not significantly negatively impacted by the bout of sleep deprivation. DISCUSSION: Some pilot-specific task-related factors such as subjective fatigue, cognitive flexibility, and working memory were found to be particularly susceptible to sleep loss, with significant declines in performance observed following 16-h continuous wakefulness, suggesting reductions in optimal functioning following this period of wakefulness. Further investigation using more regular testing time points, employing additional pilot competencies, and using more aviation-specific tasks is warranted.