Acute altitude exposure lowers arterial oxygen content ([Formula: see text]) and cardiac output ([Formula: see text]) at peak exercise, whereas O ₂ extraction from blood to working muscles remains similar. Acclimatization normalizes [Formula: see text] but not peak [Formula: see text] nor peak oxygen consumption (V̇o _2peak ). To what extent acclimatization impacts muscle O ₂ extraction remains unresolved. Twenty-one sea-level residents performed an incremental cycling exercise to exhaustion near sea level (SL), in acute (ALT1) and chronic (ALT16) hypoxia (5,260 m). Arterial blood gases, gas exchange at the mouth and oxy- (O ₂ Hb) and deoxyhemoglobin (HHb) of the vastus lateralis were recorded to assess arterial O ₂ content ([Formula: see text]), [Formula: see text], and V̇o ₂ . The HHb-V̇o ₂ slope was taken as a surrogate for muscle O ₂ extraction. During moderate-intensity exercise, HHb-V̇o ₂ slope increased to a comparable extent at ALT1 (2.13 ± 0.94) and ALT16 (2.03 ± 0.88) compared with SL (1.27 ± 0.12), indicating increased O ₂ extraction. However, the HHb/[Formula: see text] ratio increased from SL to ALT1 and then tended to go back to SL values at ALT16. During high-intensity exercise, HHb-V̇o ₂ slope reached a break point beyond which it decreased at SL and ALT1, but not at ALT16. Increased muscle O ₂ extraction during submaximal exercise was associated with decreased [Formula: see text] in acute hypoxia. The significantly greater muscle O ₂ extraction during maximal exercise in chronic hypoxia is suggestive of an O ₂ reserve.NEW & NOTEWORTHY During incremental exercise muscle deoxyhemoglobin (HHb) and oxygen consumption (V̇o ₂ ) both increase linearly, and the slope of their relationship is an indirect index of local muscle O ₂ extraction. The latter was assessed at sea level, in acute and during chronic exposure to 5,260 m. The demonstrated presence of a muscle O ₂ extraction reserve during chronic exposure is coherent with previous studies indicating both limited muscle oxidative capacity and decrease in motor drive.