Preventive effects of acetazolamide (ACZ) on acute mountain sickness (AMS) are well established but effects on exercise performance at high altitude or in hypoxia have been less considered and are still inconsistent. We hypothesized that low-dose ACZ would not impair exercise performance at simulated high altitude. Thus, the aim of this study was to evaluate the interaction between low-dose ACZ and exercise performance in normobaric hypoxia. Sixteen subjects (8 males and 8 females) were randomly assigned either to receive low-dose ACZ (3×125 mg in 36 hours) or placebo. Incremental cycle spiro-ergometry was performed before and after drug treatment in normobaric hypoxia (inspired fraction of oxygen, FiO2 = 13.5%; equivalent to about 4000 m). Whereas maximal power output and submaximal exercise responses did not change differently from pre- to post-treatment between ACZ and placebo, absolute and relative VO2max values and maximal oxygen pulse were slightly decreased in hypoxia after ACZ pre-treatment. ANOVA results suggest that aerobic capacity in males might be more affected by ACZ pre-treatment than in females. In conclusion, the presented findings may be of practical importance, possibly more meaningful for female mountaineers, because low-dose ACZ (125 mg bd) was shown to prevent AMS development with similar effectiveness as higher doses. This means that low-dose ACZ would prevent both, AMS development and a pronounced reduction in exercise performance.