Introduction: Recent evidence in isolated mitochondria and permeabilized muscle fibers in ex vivo using simultaneous measurements of O2 consumption and ATP production suggest that mitochondrial efficiency provides an additional mechanism to fine-tune oxidative phosphorylation rate to ATP demand in skeletal muscle. However, in the absence of a direct measurement of both VO2 and ATP synthesis from the same region of the contracting muscle, whether this mechanism plays a role in the skeletal muscle in vivo is still unknown. Purpose: Using a noninvasive approach combining phosphorus and proton magnetic resonance spectroscopy (31P/1H-MRS), the present study aimed to determine skeletal muscle ATP synthesis rate and muscle VO2 during a graded dynamic plantar flexion exercise to determine mitochondrial efficiency in contracting skeletal muscle contraction. Method: To measure mitochondrial efficiency under physiological conditions, we applied a recently developed methodological approach in ex vivo to human gastrocnemius muscle in vivo using 31P/1H-MRS noninvasive techniques. We conducted a series of constant workloads and ischemic protocols to assess oxidative ATP synthesis (ATPox) rate and Myoglobin-derived oxygen consumption (Mb-derived VO2). Specifically, during two separate visits, in 12 healthy, sedentary to recreationally active young male adults, we determined the ATPox rate by measuring the initial phosphocreatine (PCr) resynthesis rate during recovery and Mb-derived VO2 during 30 seconds of occlusion at the end of each given exercise workload. Results: The calculated mean power output during constant load performed by all subjects increased linearly at each incremental workload for both 1H and 31P visits. The mean percent coefficient of variation (CV%) at all exercise workloads (25% of WRmax= 6.0 ± 6.6, 50% of WRmax = 4.5 ± 2.7, 75% of WRmax = 4.9 ± 3.9, 100% = 7.0 ± 4.5) demonstrated high reliability and reproductivity in power output between each visit. The mean concentration of PCr consumption at the steady state remained constant between 25 and 50% of WRmax (28.6 ± 1.7 and 28.2 ± 1.4, respectively) but increased linearly beyond 50% of WRmax (75% = 22.9 ± 1.4 mM and 100%= 18.0 ± 1.4 mM, respectively). Mean intracellular pH was not significantly different between 25 and 50% of submaximal workloads (6.98 ± 0.02 and 6.97 ± 0.02, respectively). Intracellular pH dropped to 6.94 ± 0.02 during the last min of exercise at 75% of WRmax and further decreased at 100% of WRmax, (6.87 ± 0.03). The percentage of Mb oxygenation level and partial pressure of oxygen(PO2) at the steady state exhibited a consistent linear decline with increasing workload. Accordingly, a distinct and strong linear relationship was found between the MbO2 and workload (r2 = 0.71). Similarly, as the exercise workload increased, the ATPox synthesis rate also increased linearly throughout all exercise workloads (r2 = 0.45). In contrast, there was no significant change in Mb-derived VO2 with increased exercise workload averaged over a 30 s (r2 = 0.36) and 10 s time frame (r2 = 0.17). As a result, there was no significant correlation between ATPox synthesis rate and Mb-derived VO2 across exercise intensities. However, at rest, the P/O ratio in the gastrocnemius muscle was 1.95 ± 0.68, consistent with theoretical values and previous studies in mice. Conclusion: Despite the decrease in MbO2 (%), Mb-derived VO2 from both 30 and 10 s averages remained relatively constant during the ischemic protocol, likely due to O2 availability limitation induced by the prolonged occlusion and the slow time-resolution for measuring the dMb signal, which precluded the quantification of mitochondrial efficiency during exercise. However, mitochondrial efficiency calculated at rest was in agreement with previously documented values using other methodologies and thus can provide an additional parameter to more comprehensively evaluate mitochondrial function in vivo.