U-2 pilots routinely conduct missions in a single-seat aircraft at altitudes above 70,000 feet, requiring the pilot to wear a full pressure suit and breathe 100% oxygen. Fatigue and cognitive performance decrements are concerns in this population due to the physical and mental stresses of the U-2 environment and the prolonged mission duration of up to 12 hours. This research investigates factors that contribute to subjective fatigue and explores fatigue countermeasure usage through a paper-based questionnaire administered to deployed U-2 pilots immediately after missions. Sixty-eight completed questionnaires were retrieved during the data collection period. The same pilot may have completed multiple questionnaires for various missions. Pilots reported awakening an average of 3.07 ( 0.75) hours prior to takeoff and an average mission duration of 9.81 ( 0.48 hours), resulting in a total time awake at landing of 12.87 ( 0.94) hours. Mean mental fatigue scores (0-10 scale with 0 being not at all and 10 being extremely fatigued ) for the first half of the mission, second half of the mission, and descent and landing were reported as 4.51 ( 2.58), 4.65 ( 2.26), and 3.82 ( 2.49), respectively. Additionally, unusual physical discomfort was reported in 60.3% of missions, which may contribute to subjective fatigue. Pilots used dextroamphetamine alone or in combination with caffeine in 85.29% of the missions evaluated (n=58) to increase alertness. It appears that effective countermeasure strategies are in place to prevent increases in fatigue in prolonged U-2 missions; however, changes in mission duration, mission frequency, and/or countermeasure strategies could affect fatigue in U-2 pilots.