Abstract. Objective: Determine the relationship between caffeine, catecholamines, and alertness during prolonged wakefulness. Methods: Following 49 h of prolonged wakefulness, each of 50 healthy males (18–32 years) orally ingested either a placebo or one of three doses of caffeine, 2.1 (low), 4.3 (medium), or 8.6 mg kg–1 body weight (high), in a randomized double-blind design. Wakefulness continued for an additional 12 h during which venous blood samples were collected for catecholamine and caffeine analysis [determined using high-performance liquid chromatography (HPLC)]. A sleep latency test, the Stanford sleepiness scale, and a choice reaction time test were administered periodically during the post-dosing period and served as measures of alertness (physiological, subjective, and behavioral, respectively). Results: Caffeine had no significant effect on noradrenaline, but adrenaline was significantly increased between 1 h and 4 h post-dosing in the high dose group compared with a placebo group. Following caffeine administration, responses to sleep latency, sleepiness scores, and reaction time scores showed dose-related changes that were exhibited by significant correlation coefficients. Conclusion: The results indicate that high doses of caffeine have a significant and beneficial effect on alertness during prolonged wakefulness.