ABSTRACT: Flight crews are frequently required to work irregular schedules and, as a result, can experience sleep deficiency and fatigue. This study was conducted to determine whether perceived fatigue levels and objective performance varied by time of day, time awake, and prior night's sleep duration. Ninety-five pilots (86 male, 9 female) aged 33 years (±8) volunteered for the study. Participants completed a daily sleep diary, Samn-Perelli fatigue scale, and psychomotor vigilance task that were completed before and after each flight duty period and at the top-of-descent for each flight. Pilots experienced higher self-reported fatigue (EMM = 3.92, SE = 0.09, p < 0.001) and worse performance (Response speed: EMM = 4.27, SE = 0.08, p = 0.004) for late-finishing duties compared with early-starting duties (Samn-Perelli: EMM = 3.74, SE = 0.08; Response speed: EMM = 4.37, SE = 0.08), but had shorter sleep before early-starting duties (early: EMM = 6.94, SE = 0.10; late: EMM = 8.47, SE = 0.14, p < 0.001). However, pre-duty Samn-Perelli and response speed were worse (z = 4.18, p < 0.001; z = 3.05, p = 0.03; respectively) for early starts compared with late finishes (EMM = 2.74, SE = 0.19), while post-duty Samn-Perelli was worse for late finishes (EMM = 4.74, SE = 0.19) compared with early starts (EMM = 4.05, SE = 0.12). The results confirm that duty time has a strong influence on self-reported fatigue and performance. Thus, all flights that encroach on a biological night are targets for fatigue risk management oversight.