Purpose: Myofiber strain, Eff , is a mechanistically relevant metric of cardiac cell shortening and is expected to be spatially uniform in healthy populations, making it a prime candidate for the evaluation of local cardiomyocyte contractility. In this study, a new, efficient pipeline was proposed to combine microstructural cDTI and functional DENSE data in order to estimate Eff in vivo. Methods: Thirty healthy volunteers were scanned with three long-axis (LA) and three short-axis (SA) DENSE slices using 2D displacement encoding and one SA slice of cDTI. The total acquisition time was 11 minutes ± 3 minutes across volunteers. The pipeline first generates 3D SA displacements from all DENSE slices which are then combined with cDTI data to generate a cine of myofiber orientations and compute Eff . The precision of the post-processing pipeline was assessed using a computational phantom study. Transmural myofiber strain was compared to circumferential strain, Ecc , in healthy volunteers using a Wilcoxon sign rank test. Results: In vivo, computed Eff was found uniform transmurally compared to Ecc (-0.14[-0.15, -0.12] vs -0.18 [-0.20, -0.16], P < .001, -0.14 [-0.16, -0.12] vs -0.16 [-0.17, -0.13], P < .001 and -0.14 [-0.16, -0.12] vs Ecc_C = -0.14 [-0.15, -0.11], P = .002, Eff_C vs Ecc_C in the endo, mid, and epi layers, respectively). Conclusion: We demonstrate that it is possible to measure in vivo myofiber strain in a healthy human population in 10 minutes per subject. Myofiber strain was observed to be spatially uniform in healthy volunteers making it a potential biomarker for the evaluation of local cardiomyocyte contractility in assessing cardiovascular dysfunction.