Regional ventricular wall stress is a critical determinant of cardiac function. There are, however, no validated methods for accurately estimating this stress. We have shown in the isolated ventricular septum that, during steady-state indentations, the transverse stiffness (the ratio of indentation stress [pressure acting on indenter face] to indentation strain [amount of indentation/nonindented thickness]) can be used as an estimate of the in-plane wall stress. Because of the long acquisition time for those transverse stiffness determinations, it was not possible to follow changes in wall stress over a single contraction. We recently developed a dynamic indentation system that can determine transverse stiffness in as little as 10 ms, allowing estimation of wall stress over a single contraction cycle. The apparatus consists of an indentation probe coupled to a linear motor. This indentation system was tested on two beating canine ventricular septa that were mounted in a biaxial system the could apply strains in the plane of the septa and measure the resulting in-plane stresses. The probe indented the septa with peak displacements of 0.1-0.5 mm at frequencies of 20 and 50 Hz. The transverse stiffness was calculated as the slope of the relation between the indentation stress and indentation strain during each high-frequency indentation. Consistent with earlier studies, the transverse stiffness was related to the inplane stress. In contrast to earlier studies, however, these dynamic transverse stiffness determinations could be made during a single contraction. Thus, dynamic transverse stiffness determinations allow estimation of wall stress in the isolated septa by minimal surface contact, and may lead to methods for estimating wall stress in the intact heart.