Cast immobilization is associated with decreases in muscle contractile area, specific force and functional ability. The pathophysiological processes underlying the loss of specific force production as well as the role of metabolic alterations is not well understood. The aim of this study was to quantify changes in the resting energy rich phosphate content and specific force production following immobilization. ³¹P-Magnetic Resonance Spectroscopy, 3D-Magnetic Resonance Imaging, and isometric strength testing were performed in healthy subjects and patients with an ankle fracture following 7 weeks of immobilization and during rehabilitation. Muscle biopsies were obtained in a subset of patients. Following immobilization, there was a significant decrease in the specific plantar flexor torque and a significant increase in the inorganic phosphate (P_i) concentration (p < 0.001) as well as the ratio of P_i to phosphocreatine (P_i/PCr) (p < 0.001). No significant change in the PCr content or the basal pH values was noted. During rehabilitation, both the P_i content and P_i /PCr ratio decreased and specific torque increased, approaching control values following 10 weeks of rehabilitation. Regression analysis showed an inverse relationship between the in vivo P_i concentration and specific torque (r = 0.65, p < 0.01). In vitro force mechanics performed on skinned single human muscle fibers demonstrated that varying the P_i levels within the ranges observed across individuals in vivo (4-10 mM), changed force production by ~16 %. In summary, our findings clearly depict a change in the resting energy rich phosphate content of skeletal muscle with immobilization, which may negatively impact its force generation.