Abstract Background Multiple Acyl-CoA-Dehydrogenase deficiency (MADD) is an inherited metabolic disorder characterized by impaired oxidation of fatty acids and some amino acids. Methods We were interested whether children with MADD could tolerate a prolonged low-intensity exercise test and if this test could have any additional diagnostic value. Therefore, we performed a maximal exercise test and a low-intensity prolonged exercise test in 2 patients with MADD and in 5 control subjects. During a prolonged exercise test the subjects exercised on a cycle ergometer at a constant workload of 30% of their maximum for 90 minutes and heart rate, oxygen uptake, fuel utilization and changes in relevant blood and urinary parameters were monitored. Results The tests were tolerated well. During the prolonged exercise test the fatty acid oxidation (FAO) was quite low compared to 5 control subjects, while characteristic metabolites of MADD appeared in plasma and urine. Conclusion We suggest that the prolonged exercise test could be of diagnostic importance and might replace the fasting test as a diagnostic procedure in some cases, particularly in patients with anamnestic signs of intolerance for prolonged exercise.