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 αSMA 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 αSMA 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.