Background: Fatigue is pervasive, under-reported, and potentially deadly where flight operations are concerned. The aviation industry appears to lack a standardized, practical, and easily replicable protocol for fatigue risk assessment which can be consistently applied across operators.AimOur paper sought to present a framework, supported by real-world data with subjective and objective parameters, to monitor aircrew fatigue and performance, and to determine the safe crew configuration for commercial airline operations. Methods: Our protocol identified risk factors for fatigue-induced performance degradation as triggers for fatigue risk and performance assessment. Using both subjective and objective measurements of sleep, fatigue, and performance in the form of instruments such as the Karolinska Sleepiness Scale, Samn-Perelli Crew Status Check, Psychomotor Vigilance Task, sleep logs, and a wearable actigraph for sleep log correlation and sleep duration and quality charting, a workflow flagging fatigue-prone flight operations for risk mitigation was developed and trialed. Results: In an operational study aimed at occupational assessment of fatigue and performance in airline pilots on a three-men crew versus a four-men crew for a long-haul flight, we affirmed the technical feasibility of our proposed framework and approach, the validity of the battery of assessment instruments, and the meaningful interpretation of fatigue and work performance indicators to enable the formulation of safe work recommendations. Conclusion A standardized occupational assessment protocol like ours is useful to achieve consistency and objectivity in the occupational assessment of fatigue and work performance.