Contractile properties of myofibers are dictated by the abundance of myosin heavy chain (MyHC) isoforms. MyHC composition designates muscle function, and its alterations could unravel differential muscle involvement in muscular dystrophies and aging. Current analyses are limited to visual assessments in which myofibers expressing multiple MyHC isoforms are prone to misclassification. As a result, complex patterns and subtle alterations are unidentified. We developed a high-throughput, data-driven myofiber analysis to quantitatively describe the variations in myofibers across the muscle. We investigated alterations in myofiber composition between genotypes, 2 muscles, and 2 age groups. We show that this analysis facilitates the discovery of complex myofiber compositions and its dependency on age, muscle type, and genetic conditions.—Raz, V., Raz, Y., van de Vijver, D., Bindellini, D., van Putten, M., van den Akker, E. B. High-throughput data-driven analysis of myofiber composition reveals muscle-specific disease and age-associated patterns. FASEB J. 33, 4046–4053 (2019). www.fasebj.org.