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To cross-validate skeletal muscle oxidative capacity measured by 31P-MR spectroscopy with in vitro measurements of oxidative capacity in mitochondria isolated from muscle biopsies of the same muscle group in 18 healthy adults.Oxidative capacity in vivo was determined from PCr recovery kinetics following a 30-s maximal isometric knee extension. State 3 respiration was measured in isolated mitochondria using high-resolution respirometry. A second cohort of 10 individuals underwent two 31P-MRS testing sessions to assess the test–retest reproducibility of the method.Overall, the in vivo and in vitro methods were well-correlated (r = 0.66–0.72) and showed good agreement by Bland Altman plots. Excellent reproducibility was observed for the PCr recovery rate constant (CV = 4.6%; ICC = 0.85) and calculated oxidative capacity (CV = 3.4%; ICC = 0.83).These results indicate that 31P-MRS corresponds well with gold-standard in vitro measurements and is highly reproducible. J. Magn. Reson. Imaging 2011;. © 2011 Wiley Periodicals, Inc.