In this acute intervention study, we investigated the potential benefit of ketone supplementation in humans by studying cardiac phosphocreatine to adenosine-triphosphate ratios (PCr/ATP) and skeletal muscle PCr recovery using phosphorus magnetic resonance spectroscopy (31P-MRS) before and after ingestion of a ketone ester drink. We recruited 28 healthy individuals: 12 aged 23–70 years for cardiac 31P-MRS, and 16 aged 60–75 years for skeletal muscle 31P-MRS. Baseline and post-intervention resting cardiac and dynamic skeletal muscle 31P-MRS scans were performed in one visit, where 25 g of the ketone monoester, deltaG®, was administered after the baseline scan. Administration was timed so that post-intervention 31P-MRS would take place 30 min after deltaG® ingestion. The deltaG® ketone drink was well-tolerated by all participants. In participants who provided blood samples, post-intervention blood glucose, lactate and non-esterified fatty acid concentrations decreased significantly (−28.8%, p ≪ 0.001; −28.2%, p = 0.02; and −49.1%, p ≪ 0.001, respectively), while levels of the ketone body D-beta-hydroxybutyrate significantly increased from mean (standard deviation) 0.7 (0.3) to 4.0 (1.1) mmol/L after 30 min (p ≪ 0.001). There were no significant changes in cardiac PCr/ATP or skeletal muscle metabolic parameters between baseline and post-intervention. Acute ketone supplementation caused mild ketosis in blood, with drops in glucose, lactate, and free fatty acids; however, such changes were not associated with changes in 31P-MRS measures in the heart or in skeletal muscle. Future work may focus on the effect of longer-term ketone supplementation on tissue energetics in groups with compromised mitochondrial function.