Crew fatigue associated with successive and unaugmented 36 h missions was evaluated in B-1B simulators. Data were obtained from 32 operationally qualified crewmembers. All crew-members completed three consecutive, long duration missions, each preceded by 33 to 35 h of crew rest. Oral temperature, salivary melatonin and cortisol, as well as actigraph and subjective measures, were collected during all missions. Temperature and melatonin data indicate that crews maintained their local home base circadian cycles. Elevated cortisol and subjective fatigue during the first mission indicate that it was the most difficult of the three. Furthermore, quality and duration of sleep were lowest during the first mission. These findings emphasize the need for realistic training in long duration fatigue management to improve the safety and effectiveness of the first and subsequent missions.