Performing exercise during the first hours of hypoxic exposure is thought to exacerbate acute mountain sickness (AMS), but whether this is due to increased hypoxemia or other mechanisms associated with exercise remains unclear. In 12 healthy men, AMS symptoms were assessed during three 11-h experimental sessions: 1) in Hypoxia-exercise, inspiratory O 2 fraction (Fi O 2 ) was 0.12, and subjects performed 4-h cycling at 45% Fi O 2 -specific maximal power output from the 4th to the 8th hour; 2) in Hypoxia-rest, Fi O 2 was continuously adjusted to match the same arterial oxygen saturation as in Hypoxia-exercise, and subjects remained at rest; and 3) in Normoxia-exercise, Fi O 2 was 0.21, and subjects cycled as in Hypoxia-exercise at 45% Fi O 2 -specific maximal power output. AMS scores did not differ significantly between Hypoxia-exercise and Hypoxia-rest, while they were significantly lower in Normoxia-exercise (Lake Louise score: 5.5 ± 2.1, 4.4 ± 2.4, and 2.3 ± 1.5, and cerebral Environmental Symptom Questionnaire: 1.2 ± 0.7, 1.0 ± 1.0, and 0.3 ± 0.4, in Hypoxia-exercise, Hypoxia-rest, and Normoxia-exercise, respectively; P < 0.01). Headache scored by visual analog scale was higher in Hypoxia-exercise and Hypoxia-rest compared with Normoxia-exercise (36 ± 22, 35 ± 25, and 5 ± 6, P < 0.001), while the perception of fatigue was higher in Hypoxia-exercise compared with Hypoxia-rest (60 ± 24, 32 ± 22, and 46 ± 23, in Hypoxia-exercise, Hypoxia-rest, and Normoxia-exercise, respectively; P < 0.01). Despite significant physiological stress during hypoxic exercise and some AMS symptoms induced by normoxic cycling at similar relative workload, exercise does not significantly worsen AMS severity during the first hours of hypoxic exposure at a given arterial oxygen desaturation. Hypoxemia per se appears, therefore, to be the main mechanism underlying AMS, whether or not exercise is performed.