Complex I Deficiency (CID) is a disorder of oxidative phosphorylation characterized by impaired mitochondrial ability to oxidize NADH. Triglyceride compared to glucose oxidation provides a higher non-NADH vs. NADH electron flow to the respiratory chain; we thus postulated that supplying more fat would improve exercise tolerance and oxygen consumption in CID patients. In 3 CID patients with decreased maximal workload (mean (Wmax; mean (SD) 53 (7) W) and in 3 control subjects (Wmax 197 (15) W), we studied the effects of intravenous infusion of isoenergetic amounts of Intralipid (3.6 mg triglyceride/kg/min) or glucose (10 mg/kg/min) during steady-state bicycle ergometry at 15% Wmax. Results showed that the exercise was tolerated for the nominal 90 minutes in all control studies, and also in the CID patients during triglyceride infusion. However, during glucose infusion all CID patients suffered from premature muscle fatigue (mean endurance time 58 (95% CI: 36-80) min). Oxygen consumption in the CID patients during cycling was higher with triglyceride infusion compared to glucose infusion (difference: 1.0 (95% CI: 0.4-1.5) ml/kg/min); in the controls no significant difference was found (0.1(-0.2 to 0.4) ml/kg/min). In the exercising CID patients (but not in controls), plasma lactate concentrations rose substantially (with glucose to 8.6 (5.0) mM, with triglyceride to 8.9 (2.8) mM; infusion effect not significant) above baseline. Effects of high fat/low carbohydrate vs. high carbohydrate/low fat diets (Protein: 15 energy%; Fat: 55 vs. 25 energy%, respectively) were studied in the CID patients in a cross-over design, during at least 2 weeks for each dietary period. Steady-state bicycle ergometry at 15% of initial Wmax showed reduced endurance time on the low fat/high carbohydrate diet in two (28 and 23 min) but normal endurance (90 min) in one patient. Endurance time on the high fat diet was 90 min in all patients. We conclude that fat as compared to carbohydrates improves mitochondrial performance in affected muscle in patients with Complex I Deficiency. The results suggest the preferential use of fat, or avoidance of carbohydrate-rich meals before exercise, by patients with this respiratory chain disorder.