Introduction: Fatigue is an acknowledged safety risk in diverse operational settings. As a result, there has been growing interest in developing and implementing activities to improve alertness, performance, and safety in real-world operations where fatigue is a factor. Methods: A comprehensive Alertness Management Program (AMP) that included education, alertness strategies, scheduling, and healthy sleep was implemented in a commercial airline. An operational evaluation was conducted with 29 flight crewmembers, first when flying a standard schedule without AMP components (i.e., standard condition) compared with full AMP implementation, which included flying an innovative schedule that incorporated physiological sleep and alertness principles (i.e., intervention condition). The evaluation included objective measures of sleep quantity (actigraphy), psychomotor vigilance task (PVT) performance, and subjective reports of daily activities and sleep. Results: The results showed that the 3.5-h educational CD improved pre-education test scores from an average 74% correct to a post-education average of 98%. Alertness strategies showed minimal changes, though the daily diary did not allow for refined evaluation of duration, frequency, and timing of use. The intervention condition was associated with significantly more sleep (1 h, 9 min; p < 0.01) during the trip period compared with the standard schedule. All performance metrics showed significantly better performance during the intervention condition trip schedule (p < 0.01) compared with the standard condition. Discussion: This first-ever evaluation of a comprehensive AMP showed significantly improved knowledge, support for the use of alertness strategies, and increased sleep and performance during actual operations. The robust and consistent findings support the use of an AMP approach to effectively manage fatigue in operational settings.