Aim Our aim was to investigate if there are differences in mitochondrial energy metabolism in working quadriceps and calves muscles by dynamic localized phosphorus-31 magnetic resonance spectroscopy. Materials and methods Phosphate metabolites of muscles were detected while subjects were in a state of rest, during exercise and recovery. The phosphocreatine (PCr), inorganic phosphate (Pi), adenosine triphosphate (ATP), PCr/Pi, PCr/ATP, pH, work/energy cost ratio (WE) and oxidative capacity were compared between muscles. Results The quadriceps had larger volume, heavier exercise load, greater WE, lower PCr and Pi concentration than calves muscles at rest. The PCr/Pi ratio of both muscles exhibited a sharp decline during exercise. PCr/ATP also had a downtrend in during exercise. Meanwhile, PCr/ATP of quadriceps during exercise was lower than that of calves muscles. The ATP concentration and pH value of quadriceps were decreased in end exercise compared to rest. And our results indicated that quadriceps had higher energy transfer efficiency and relatively poor energy buffering capability than calves muscles. The change in log(PCr) could adopt a linear fit model for both muscles at recovery. Conclusion Quadriceps had higher energy transfer efficiency and relatively poor energy buffering capability than calves muscles in mitochondrial energy metabolism partially accounts for different fiber type compositions.