The cardiomyocyte cytoskeleton, composed of rigid and elastic elements, maintains the shape of an elongated cylinder with an eccentrically-ellipsoidal cross-section, even during contraction-relaxation cycles. Mitochondria are micron-sized fluid-filled passive spheres distributed throughout the cardiomyocyte in a crystal-like lattice in pairs sandwiched between the sarcomere contractile machinery, longitudinally and radially; thus, their shape represents the balance of forces in 3D extant at any given moment. We developed a novel method to examine the average deformation of mitochondrial dimensions in 3D, in response to cardiomyocyte contraction and relaxation, to understand how dynamic forces are balanced inside the cardiomyocytes. The optical contrast provided by the periodic lattice of myofilaments alternating with rows of mitochondria was analyzed by examining the appropriate peaks in the frequency spectrum image along the respective cardiomyocyte axes. This technique enables precise resolution of changes in dimension of ∼1% in ∼1 µm (long axis) structures with a time resolution of 8 msec.During active contraction (1 Hz stimulation) the mitochondria deform along the length-and width-axes with similar time-to-peak deformation and 50% and 90% deformation duration characteristics in both sarcomere and mitochondrial structures. However, significant deformation anisotropy was observed between the orthogonal short (i.e., width & depth) axes of mitochondria during electrical stimulation. Interestingly, the same degree of deformation anisotropy was found between the myocyte orthogonal short axes during the same electrical stimulation; therefore, the mitochondria reflect the overall cell behavior, and the apparent stiffness and stress/strain characteristics of the cytoskeleton differ appreciably between the cardiomyocyte orthogonal short axes. This method may be applied to obtaining a better understanding of the dynamic force-balance inside cardiomyocytes and of changes in the cytoskeleton spatial stiffness characteristics that may accompany aging or pathological conditions.