Key PointsQuestionDoes mitochondrial dysfunction underlie altered skeletal muscle metabolism and exercise intolerance in patients with heart failure with preserved ejection fraction (HFpEF)?FindingsIn this cross-sectional study including 27 patients older than 60 years with HFpEF and 45 healthy age-matched controls, high-resolution respirometry of vastus lateralis muscles from patients with HFpEF revealed markedly reduced bioenergetic capacity associated with peak exercise oxygen consumption and exercise performance (6-minute walk distance, Short Physical Performance Battery, and leg strength).MeaningIn this study, detailed analysis of mitochondrial function provided evidence that skeletal muscle mitochondrial dysfunction can play a role in HFpEF exercise intolerance, which may impact the development of therapeutic strategies that target mitochondrial dysfunction in patients with HFpEF.