Changes in left ventricular (LV) aggregate cardiomyocyte orientation and deformation underlie cardiac function and dysfunction. As such, in vivo aggregate cardiomyocyte "myofiber" strain ( [Formula: see text]) has mechanistic significance, but currently there exists no established technique to measure in vivo [Formula: see text]. The objective of this work is to describe and validate a pipeline to compute in vivo [Formula: see text] from magnetic resonance imaging (MRI) data. Our pipeline integrates LV motion from multi-slice Displacement ENcoding with Stimulated Echoes (DENSE) MRI with in vivo LV microstructure from cardiac Diffusion Tensor Imaging (cDTI) data. The proposed pipeline is validated using an analytical deforming heart-like phantom. The phantom is used to evaluate 3D cardiac strains computed from a widely available, open-source DENSE Image Analysis Tool. Phantom evaluation showed that a DENSE MRI signal-to-noise ratio (SNR) ≥20 is required to compute [Formula: see text] with near-zero median strain bias and within a strain tolerance of 0.06. Circumferential and longitudinal strains are also accurately measured under the same SNR requirements, however, radial strain exhibits a median epicardial bias of -0.10 even in noise-free DENSE data. The validated framework is applied to experimental DENSE MRI and cDTI data acquired in eight ( N=8 ) healthy swine. The experimental study demonstrated that [Formula: see text] has decreased transmural variability compared to radial and circumferential strains. The spatial uniformity and mechanistic significance of in vivo [Formula: see text] make it a compelling candidate for characterization and early detection of cardiac dysfunction.