Abstract Background and Aims Patients with heart failure and preserved ejection fraction (HFpEF) experience significant exercise intolerance, yet its underlying mechanisms remain poorly defined and are multifactorial. Iron deficiency (ID) occurs frequently in HFpEF and may contribute to exercise impairment. This study evaluated mitochondrial oxidative muscle metabolism in HFpEF using in vivo 31phosphorus magnetic resonance spectroscopy (31P-MRS) employing two exercise protocols, and assessed whether ID influences exercise energetics. Methods In this parallel analysis of prospective studies at two sites, patients with HFpEF and control individuals performed either isometric exercise (isolated leg protocol) or dynamic exercise (cardiopulmonary protocol) with concomitant phosphocreatine recovery assessment using in vivo 31P-MRS. Associations between clinical factors and oxidative metabolism were evaluated. ID was defined as ferritin <100 µg/L, or ferritin 100–299 µg/L with transferrin saturation <20%. Results Fifty-eight patients with HFpEF and 16 controls performed isometric exercise (n = 46 HFpEF; n = 16 control) or cardiopulmonary exercise (n = 12 HFpEF). Phosphocreatine recovery halftime after isometric exercise was prolonged in patients versus controls [27 (23–32) vs 24 (19–28) seconds, respectively; P = .03]. Phosphocreatine recovery halftime after dynamic exercise in patients was 39 (27–57) seconds. Both cohorts consisted of patients with and without ID (n = 19 and 27, and n = 6 and 6, respectively), who had comparable exercise and oxidative muscle capacity (all P > .42). High-sensitive C-reactive protein was associated with prolonged phosphocreatine recovery halftime (P =. 01). Conclusions Patients with HFpEF exhibit impaired whole-muscle oxidative capacity of skeletal muscle, as shown by two different 31P-MRS protocols with upper leg measurements, independent of ID status. Study registration NTR6605, NTR7297 (https://onderzoekmetmensen.nl/nl/trial/55673); NCT05750940 (https://clinicaltrials.gov/study/NCT05750940).