INTRODUCTION: Fatigue is a major contributor to aviation accidents. Sufficient sleep may be difficult to achieve under operational conditions in military aviation. Countermeasures include caffeine, however, studies evaluating its effects often do not represent daily practice with regular caffeine consumption. This study aims to establish the effect of caffeine on psychomotor performance in a realistic scenario (i.e., after a limited period of extended wakefulness).METHODS: This randomized, double-blind, crossover, placebo-controlled trial included 30 aeromedically fit subjects. On trial days, subjects followed their normal routine till 17:00, after which caffeine intake was stopped. At midnight, subjects were given 300 mg of caffeine or placebo and performed the Psychomotor Vigilance Test, Vigilance and Tracking Test, and the Stanford Sleepiness Scale hourly up to 04:00 and again at 06:00 and 08:00. Four blood samples were collected. Statistical analyses included repeated-measures ANOVA or Friedman tests, marginal models, and Wilcoxon Signed Rank tests.RESULTS: Median time awake at midnight was 17 h (IQR 16.5–17.5 h). Performance decreased significantly less during the night in the caffeine condition versus placebo. Neither habitual intake nor daytime caffeine consumption affected this. No statistically significant correlation was identified between blood concentrations of caffeine and performance.DISCUSSION: A single dose of 300 mg of caffeine has beneficial effects on performance during the night in a realistic scenario for military aviation. Daytime caffeine consumption does not affect the effects of caffeine at night. These findings could be relevant for all industries in which optimal performance is required during nighttime after a limited period of extended wakefulness.Wingelaar-Jagt YQ, Wingelaar TT, de Vrijer L, Riedel WJ, Ramaekers JG. Daily caffeine intake and the effect of caffeine on pilots’ performance after extended wakefulness. Aerosp Med Hum Perform. 2023; 94(10):750–760.