The respective oxidation of glycerol and glucose (0.36 g/kg each) ingested simultaneously immediately before exercise (120 min at 68 ± 2% maximal oxygen uptake) was measured in six subjects using ¹³C labeling. Indirect respiratory calorimetry corrected for protein and glycerol oxidation was used to evaluate the effect of glucose + glycerol ingestion on the oxidation of glucose and fat. Over the last 80 min of exercise, 10.0 ± 0.8 g of exogenous glycerol were oxidized (43% of the load), while exogenous glucose oxidation was 21% higher (12.1 ± 0.7 g or 52% of the load). However, because the energy potential of glycerol is 18% higher than that of glucose (4.57 vs. 3.87 kcal/g), the contribution of both exogenous substrates to the energy yield was similar (4.0-4.1%). Total glucose and fat oxidation were similar in the placebo (144.4 ± 13.0 and 60.5 ± 4.2 g, respectively) and the glucose + glycerol (135.2 ± 12.0 and 59.4 ± 6.5 g, respectively) trials, whereas endogenous glucose oxidation was significantly lower than in the placebo trial (123.7 ± 11.7 vs. 144.4 ± 13.0 g). These results indicate that exogenous glycerol can be oxidized during prolonged exercise, presumably following conversion into glucose in the liver, although direct oxidation in peripheral tissues cannot be ruled out.