Due to presumed adverse performance impact, a World Health Organization clause currently restricts the use of mefloquine malaria chemoprophylaxis in individuals requiring fine coordination and spatial discrimination. We conducted a double-blind, placebo-controlled, cross-over study to quantitatively assess the effects of mefloquine at steady state on performance in 23 trainee airline pilots. Flying performance was assessed using a flight simulator, psychomotor function was evaluated, sleep and wake cycles were monitored, and symptoms and moods were assessed using standardized questionnaires. A simplified postural sway meter recorded sway in three test positions. In the mefloquine loading dose phase, there was one withdrawal due to dizziness, diarrhea, and flu-like symptoms, and three volunteers reported nonserious, sleep-related adverse events. There was no significant difference in flying performance, psychomotor functions, or mean sway for any test position. Nonsignificant reductions in mean total nocturnal sleep (mefloquine = 450 min versus placebo = 484 min) and poorer sleep quality were detected in the mefloquine phases. The mood findings indicated a predominance of positive states, with vigor the predominant mood in all phases. No significant performance deficit was documented under laboratory conditions during use of mefloquine at steady state.