Abstract Introduction Prior simulation and operational studies have started to address whether the number of consecutive flight segments negatively affects cognitive performance, fatigue, and sleepiness, without reaching a clear consensus. This study expands this literature by determining whether there are significant changes in cognitive performance, fatigue, and sleepiness at critical phases of flight across multiple flight segments, while accounting for the number of segments, flight direction, trip day, and time-of-day. Methods Fifty commercial airline pilots were studied. Each pilot flew two separate short-haul trips, each ranging from 1–4 days and 1–10 flight segments. Cognitive performance, fatigue, and sleepiness were assessed at top-of-climb (TOC) and top-of-descent (TOD) of each flight segment and each trip day. Cognitive performance, fatigue, and sleepiness were assessed using Psychomotor Vigilance Task (PVT) speed, Samn-Perelli (SP) ratings, and Karolinska Sleepiness Scale (KSS) ratings, respectively. Data were analyzed using Wilcoxon t-tests and verified using ANOVAs. Results Mean PVT speed (Cohen’s d =0.57), SP ratings (Cohen’s d = 0.73), and KSS ratings (Cohen’s d = 0.63) were significantly worse at TOD than TOC (p < 0.001); and, significantly varied across flight segments (p<0.001). Cognitive performance, fatigue, and sleepiness were consistently and significantly degraded around the fifth flight segment, improved around the sixth to eighth flights segments, and were subsequently degraded around the eighth to tenth flight segments. Conclusion The results indicate that cognitive performance, fatigue, and sleepiness vary across flight segments, trip day, and phase of flight. Results suggest that these safety performance indices degrade after five segments, and further degrade after eight flight segments. The results presented could be used to inform future airline scheduling and regulation. Support This work has been supported by United Airlines.