Purpose: To study proton magnetic resonance spectra (1H-MRS) of the muscle metabolite of a leg muscle in neutral (NEU), internal rotation (INT), and external rotation (EXT) leg positioning. Material and methods: The volunteers were selected for this study. The tibialis anterior (TA), soleus (SOL), and gastrocnemius (GAS) muscles of a non-dominate leg were determined by using single-voxel spectroscopy 8 × 8 × 20 mm3 in size. 1H-MRS measurements were performed on a 1.5-Tesla magnetic resonance imaging (MRI) scanner. Results: The results showed that metabolite spectrum of muscle in each NEU, INT, and EXT of leg positioning were not similar. Additionally, the quantification of IMCL (CH3) and EMCL (CH3) is significantly different in SOL. Conclusions: Our study showed that leg positioning influences the appearance and quantification of 1H-MRS in the calf muscle. Hence, it is necessary to pay close attention to positioning because it interferes with spectral fitting and quantification.