Cardiomyocytes both cause and experience continuous cyclic deformation, with up to ±10% changes in length compared to their resting state. The 3 dimensional (3D) organization of intracellular structures will change, in this context, on a beat-by-beat basis – but the exact effects of mechanical deformation on the topology of intracellular organelles are not well characterised. Cardiomyocytes have low free cytosolic volume. Intracellular organelles (such as T-tubules, sarcoplasmic reticulum or mitochondria) are arranged between strands of the contractile filament lattice, and embedded within a network of rigid and elastic non-sarcomeric cytoskeletal elements that bear and transmit mechanical loads throughout the cell. We investigated deformation-induced 3D nano-scale changes in cardiomyocyte ultrastructure, with focus not only on individual organelles, but also on spatial relationships between homo- and heterotypic structures. To determine how mechanical cell activity alters the 3D nano-topography of (primarily membranous) cell compartments, we utilise electron tomography of rabbit cardiac tissue and of isolated cells. Furthermore, using live-cell imaging, we assessed the functional impact of beat-by-beat structural alterations on dynamic intracellular processes such T-tubular content exchange dynamics. Key findings are that T-tubules deform in a sarcomere-length dependent manner, without notable effects on intra-tubular diffusion. The results presented here provide reference data for spatial contextualisation of functionally-relevant ultrastructures and their mechanical modulation in healthy cardiomyocytes.