Barth syndrome (BTHS) is a mitochondrial myopathy characterized by reports of exercise intolerance. We sought to determine if 1) BTHS leads to abnormalities of skeletal muscle O2 extraction/utilization and 2) exercise intolerance in BTHS is related to impaired O2 extraction/utilization, impaired cardiac function, or both. Participants with BTHS (age: 17 ± 5 yr, n = 15) and control participants (age: 13 ± 4 yr, n = 9) underwent graded exercise testing on a cycle ergometer with continuous ECG and metabolic measurements. Echocardiography was performed at rest and at peak exercise. Near-infrared spectroscopy of the vastus lateralis muscle was continuously recorded for measurements of skeletal muscle O2 extraction. Adjusting for age, peak O2 consumption (16.5 ± 4.0 vs. 39.5 ± 12.3 ml·kg−1·min−1, P < 0.001) and peak work rate (58 ± 19 vs. 166 ± 60 W, P < 0.001) were significantly lower in BTHS than control participants. The percent increase from rest to peak exercise in ejection fraction (BTHS: 3 ± 10 vs. control: 19 ± 4%, P < 0.01) was blunted in BTHS compared with control participants. The muscle tissue O2 saturation change from rest to peak exercise was paradoxically opposite (BTHS: 8 ± 16 vs. control: −5 ± 9, P < 0.01), and the deoxyhemoglobin change was blunted (BTHS: 0 ± 12 vs. control: 10 ± 8, P < 0.09) in BTHS compared with control participants, indicating impaired skeletal muscle extraction in BTHS. In conclusion, severe exercise intolerance in BTHS is due to both cardiac and skeletal muscle impairments that are consistent with cardiac and skeletal mitochondrial myopathy. These findings provide further insight to the pathophysiology of BTHS.