© 2015 The Association for Research in Vision and Ophthalmology, Inc. PURPOSE. To determine the effects of altered mechanical strain on human peripapillary scleral (ppSc) fibroblast-to-myofibroblast differentiation. METHODS. Eight human ppSc fibroblast cultures were isolated from three paired eyes and two unilateral eyes of five donors using an explant approach. Human ppSc fibroblast isolates were subjected to 1% and 4% cyclic strain at 0.05 to 5 Hz for 24 hours. Levels of α smooth muscle actin (αSMA) mRNA and protein were determined by real-time PCR and immunoblot. Incorporation of αSMA into actin stress fibers was evaluated by confocal immunofluorescent microscopy. Myofibroblast contractility was measured by fibroblast-populated three-dimensional collagen gel contraction assay and phosphorylation of myosin light chain (MLC20). RESULTS. Human ppSc fibroblasts contained 6% to 47% fully differentiated myofibroblasts before strain application; 4% cyclic strain increased aSMA mRNA and protein expression in ppSc fibroblasts compared with 1% strain applied at 5 Hz, but not at lower frequencies. Seven of eight ppSc fibroblast isolates responded to high-magnitude and high-frequency strain with increased cellular contractility and increased MLC20 phosphorylation. In addition, increasing strain frequency promoted aSMA expression in ppSc fibroblasts under both 1% and 4% strain conditions. CONCLUSIONS. High-magnitude and/or high-frequency mechanical strain promotes differentiation of human ppSc fibroblasts into contractile myofibroblasts, a fibroblast phenotypic change known to be key to tissue injury-repair responses. These findings suggest that the cellular constituent of ppSc may play an important role in the regulation of optic nerve head biomechanics in response to injurious IOP fluctuations.