Myocardial stiffness exhibits cyclic variations over the course of the cardiac cycle. These trends are closely tied to the electro-mechanical and hemodynamic changes in the heart. Characterization of dynamic myocardial stiffness can provide insights into the functional state of the myocardium as well as allow for differentiation between the underlying physiological mechanisms that lead to congestive heart failure. Previous work has demonstrated the potential of acoustic radiation force impulse (ARFI) imaging to capture temporal trends of myocardial stiffness in experimental preparations such as the Langendorff heart as well as on animals in open-chest and intracardiac settings. This study aims to investigate the potential of ARFI to measure dynamic myocardial stiffness on human subjects, in a noninvasive manner through transthoracic imaging windows. ARFI imaging was performed on twelve healthy volunteers to track stiffness changes within the interventricular septum (IVS) in the parasternal long-axis (PLAX) and short-axis (PSAX) views. Myocardial stiffness dynamics over the cardiac cycle were quantified using five indices: stiffness ratio, rates of relaxation and contraction, and time constants of relaxation and contraction. Yield of ARFI acquisitions was evaluated based on metrics of signal strength and tracking fidelity such as displacement signal-to-noise, signal-to-clutter level, temporal coherence of speckle, and spatial similarity within the region-of-excitation (ROE). These were quantified using the mean ARFinduced displacements over the cardiac cycle, the contrast between the myocardium and the cardiac chambers, the minimum correlation coefficients of RF signals, and the correlation between displacement traces across simultaneously-acquired azimuthal beams, respectively.