We investigated the in vivo skeletal muscle metabolism in patients with multiple acyl-CoA dehydrogenase deficiency (MADD) during exercise, and the effect of a glucose infusion. Two adults with MADD on riboflavin and l-carnitine treatment and 10 healthy controls performed an incremental exercise test measuring maximal oxidative capacity (VO2max) and a submaximal exercise test (≤1 hour) on a cycle ergometer. During submaximal exercise, we studied fat and carbohydrate oxidation, using stable isotope tracer methodology and indirect calorimetry. On another day, the patients repeated the submaximal exercise receiving a 10% glucose infusion. The patients had a lower VO2max than controls and stopped the submaximal exercise test at 51 and 58 minutes due to muscle pain and exhaustion. The exercise-induced increase in total fatty acid oxidation was blunted in the patients (7.1 and 1.1 vs 12 ± 4 μmol × kg-1 × min-1 in the healthy controls), but total carbohydrate oxidation was higher (67 and 63 vs 25 ± 11 μmol × kg-1 × min-1 in controls). With glucose infusion, muscle pain decreased and average heart rate during exercise dropped in both patients from 124 to 119 bpm and 138 to 119 bpm. We demonstrate that exercise intolerance in MADD-patients relates to an inability to increase fat oxidation appropriately during exercise, which is compensated partially by an increase in carbohydrate metabolism.