**Background:** Heart failure with preserved ejection fraction (HFpEF) is the most prevalent form of heart failure, particularly among older adults and women. The primary clinical manifestation is severe exercise intolerance, measured as reduced peak oxygen uptake (peak VO2). While cardiac dysfunction contributes, prior evidence suggests that noncardiac peripheral factors—particularly skeletal muscle abnormalities—play a major role. Previous studies using frozen tissue samples showed reduced mitochondrial content and oxidative capacity in HFpEF skeletal muscle, but direct respirometric analysis of mitochondrial function in fresh tissue had not been performed. This study aimed to provide the first direct assessment of mitochondrial function using high-resolution respirometry in freshly obtained skeletal muscle from patients with HFpEF.
**Methods:** This cross-sectional study included 27 patients with HFpEF (mean age 68.4 years, 85% women, mean BMI 38.9) and 45 age-matched healthy controls (mean age 70.2 years, 80% women, mean BMI 26.8). HFpEF was defined per 2013 ACC/AHA guidelines (ejection fraction ≥50%, diastolic dysfunction grade ≥1, BMI ≥28). Participants underwent cardiopulmonary exercise testing (modified Naughton protocol for HFpEF, modified Bruce for controls), 6-minute walk test, Short Physical Performance Battery (SPPB), and leg strength testing via dynamometry. Vastus lateralis muscle biopsies were obtained after an overnight fast. High-resolution respirometry was performed on permeabilized muscle fiber bundles using an Oroboros Oxygraph-2k, measuring oxygen consumption across complex I respiration, complexes I and II respiration, and maximal uncoupled capacity. Statistical analyses included independent-samples t-tests, χ² tests, ANCOVA adjusting for sex, age, and BMI, and Pearson correlations.
**Key Results:** Patients with HFpEF had severely reduced peak VO2 compared with controls (14.8 vs 26.0 mL/kg/min, p<0.001). Six-minute walk distance was significantly lower (373 vs 546 m, p<0.001), SPPB total score was lower (9.9 vs 11.6 units, p<0.001), and left leg strength was reduced (79.9 vs 107.7 N·m, p=0.006). Skeletal muscle mitochondrial respiration was markedly reduced in HFpEF across all measures: complex I respiration (10.7 vs 28.2 pmol/s/mg, p<0.001), complexes I and II respiration (15.9 vs 40.1 pmol/s/mg, p<0.001), and maximal capacity (24.4 vs 61.4 pmol/s/mg, p<0.001). All differences remained statistically significant after adjustment for sex, age, and BMI individually and together (all p<0.001). Significant positive correlations were found between mitochondrial respiration and peak VO2 (R=0.70 for complex I, R=0.69 for complexes I+II, R=0.69 for maximal capacity; all p<0.001), 6-minute walk distance (R=0.69, p<0.001), leg strength (R=0.41, p<0.001), and SPPB scores (R=0.46, p<0.001). Sensitivity analyses showed similar correlations within HFpEF (R=0.28, p=0.15) and HC groups (R=0.28, p=0.06) for peak VO2 and maximal capacity, with nonsignificant interaction tests.
**Clinical Implications:** This study provides the first direct evidence that skeletal muscle mitochondrial dysfunction is present in patients with HFpEF and is strongly associated with multiple objective measures of exercise intolerance. The findings suggest that mitochondrial abnormalities represent a potential therapeutic target, as mitochondrial function is modifiable through both pharmacological interventions (e.g., Szeto-Schiller peptides, coenzyme Q10, MitoQ, nicotinamide mononucleotide) and behavioral interventions such as exercise training. The data support the concept that noncardiac peripheral factors, particularly skeletal muscle metabolism, contribute significantly to exercise intolerance in HFpEF. Limitations include the higher BMI in HFpEF patients (reflecting the typical HFpEF phenotype), potential differences in physical activity levels not measured, and the cross-sectional design which cannot establish causality. Future studies should examine whether interventions targeting mitochondrial function can improve exercise capacity in patients with HFpEF.