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ABSTRACT To investigate the effect of acclimatization to hypoxia on substrate utilization, eight sea level residents were studied during exercise at the same relative (rel) and absolute (abs) work rate as at sea level (SL), under acute (AH), and after 4 weeks exposure to 4100 m altitude (CH). Carbohydrate (CHO) and fat oxidation during exercise at SL were 2.0 ± 0.2 and 0.3 ± 0.0 g min−1, respectively. At AHabs and CHabs CHO oxidation increased (P < 0.05) to 2.5 ± 0.2 and 2.3 ± 0.1 for CHO, and fat oxidation decreased (P < 0.05) to 0.2 ± 0.01 and 0.2 ± 0.01 g min−1, respectively. Exercise in AHrel and CHrel did not cause a change in the relative CHO and fat oxidation compared with SL, the absolute rate of CHO oxidized being 1.7 ± 0.1 and 1.7 ± 0.02 g min−1, respectively, and fat oxidation was 0.2 ± 0.02 g min−1 in ACrel and 0.3 ± 0.02 g min−1 in CHrel. In conclusion, substrate utilization is unaffected by AH and CH, when the work rate is matched to the same relative intensity as at SL.