Aims: To evaluate the feasibility of quantitative analysis of muscle stiffness by Acoustic Radiation Forced Impulse (ARFI) elastography over a course of graduate isometric voluntary contractions. Material and methods: The stiffness of the bilateral biceps muscle of 13 healthy volunteers was measured in real time by ARFI elastography, while the forearm was in neutral-extended position, 90 degree self-flexed positions and 90 degree self-flexed position, with altered weights ranging from 1 to 8 kg placed on flattened palmar surfaces consecutively. The determined increases in biceps muscle stiffness were measured for both arms and correlated with the loadings weights adopted at progressive trial stages. Results: The mean shear wave velocity (SWV) values of biceps muscles in a neutral position, in 90 degree flex position and 90 degree flex position with 1 to 8 kg weights on palmar surfaces were 2.162 +/- 0.302 m/sec, 3.382 +/- 0.581 m/sec, and 3.897 +/- 0.585 to 5.562 +/- 0.587 m/sec, respectively. Significant correlations between the muscle SWV values and related palmar weights and between the SWV values of right and left sides at different trial stages were identified (r=0.951 and r=0.954, respectively). A mutual propagation path of deep regions to entire areas was described to account for the distribution of increase in stiffness with increases in palmar weights. The confidence of method regarding inter-observer difference was confirmed by the correlation analyses of the results (r=0.998). Conclusions: ARFI elastography is a feasible imaging modality for quantifying the stiffness of isometrically voluntarily contracting muscles.