Purpose We aimed to investigate dynamic changes of phosphate metabolite levels in exercising calf muscle in healthy young volunteers using 31P Magnetic Resonance Spectroscopy (31P-MRS) during exercise controlled by a custom built quantitative system, and to investigate the difference between male and female. Methods A total of 19 healthy volunteers (11 male and 8 female), male aged 21-25 yr (mean 23.5 ± 1.3 yr) and female aged 22-25 yr (mean 23.8 ± 0.9 yr), performed plantar flexion excise while lying supine in Philips 3T Magnetic Resonance (MR) scanner (Achieva 3.0T TX, best, the Netherlands) using custom built exercise system. The 31P-MRS coil is firmly placed under the calf muscle. 31P-MRS measurements of inorganic phosphate (Pi), phosphocreatine (PCr) and adenosine triphosphate (ATP) were acquired from calf muscle while subjects were in a state of pre-, during and post-exercise. The Pi/PCr, β-ATP/PCr, ATP/PCr, PH and the male and female work efficiency were calculated respectively. The work efficiency depends on the ratio of the load to Pi/PCr. Results There were no statistically significance differences in age and body mass index (BMI) between men and women. But, the male loads were higher than women (P<0.05). There were no differences in ATP/PCr, β-ATP/PCr and PH in men and women between different phases. But the Pi/PCr significantly increased during exercise compared with pre- and post-exercise both in men and women (P<0.001). And, the male Pi/PCr was significantly lower than that of women during exercise (P=0.023). The male work efficiency was significantly higher than women (P=0.001). The PH increased during exercise and rapidly decreased postexercise compared with pre-exercise both in men and women (P<0.001). Conclusions The changing of Pi/PCr and work efficiency could be used as a marker for monitoring muscle mitochondria metabolism. This study showed promising results by using an in-house built MR compatible quantitative force control system with feedback mechanism.