The value of quantitative MR methods as potential biomarkers in neuromuscular disease is being increasingly recognized. Previous studies of the magnetization transfer ratio have demonstrated sensitivity to muscle disease. The aim of this work was to investigate quantitative magnetization transfer imaging of skeletal muscle in healthy subjects at 3 T to evaluate its potential use in pathological muscle. The lower limb of 10 subjects was imaged using a 3D fast low-angle shot acquisition with variable magnetization transfer saturation pulse frequencies and amplitudes. The data were analyzed with an established quantitative two-pool model of magnetization transfer. T(1) and B(1) amplitude of excitation radiofrequency field maps were acquired and used as inputs to the quantitative magnetization transfer model, allowing properties of the free and restricted proton pools in muscle to be evaluated in seven different muscles in a region of interest analysis. The average restricted pool T(2) relaxation time was found to be 5.9 ± 0.2 μs in the soleus muscle and the restricted proton pool fraction was 8 ± 1%. Quantitative magnetization transfer imaging of muscle offers potential new biomarkers in muscle disease within a clinically feasible scan time.