PURPOSE. Cyclic stretching of the glial fibrillary acidic protein (GFAP)-negative lamina cribrosa (LC) cell in vitro is associated with transcriptomic changes in genes involved in extracellular matrix (ECM) dynamics in vivo, thereby implicating this cell type in the pathophysiologic changes of the optic nerve head (ONH) in glaucoma. The purpose of the study was to determine whether exposure to different grades of mechanical stretch progressively alters the expression of ECM genes in cultured LC cells. METHODS. Primary cultures of human LC cells from three separate donors were maintained in static culture or exposed to low-level strain (3% ±0.5% elongation, 1 Hz) for 24 hours. A baseline comparison of the expression of 62 genes involved in ECM dynamics was performed with low-density gene arrays (LDAs). The 3% protocol was used in a 24-hour period of baseline dynamic low-level stretch, and gene expression was compared with that occurring in a further 24-hour exposure to a 12.5% or a 20% stretch. Gene expression levels were determined by qRT-PCR. RESULTS. LC cells displayed a nonlinear, transcriptional response to the mechanical stretch. Ten ECM-related and growth factor genes were altered by 3% strain versus static culture (nine downregulated and one upregulated). Increasing strain from 3% to 20% resulted in a significant increase in expression of 15 ECM-elated genes. Only one gene (epidermal growth factor) was increased between the 3% and 12.5% strains. CONCLUSIONS. Low-level, pulsatile, cyclic strain resets a lower baseline expression of several glaucoma-associated ECM genes. The LC ECM gene response occurs above a fourfold increase in baseline strain (12.5% strain) in vitro. The study supports the use of a nonstatic baseline when studying the effect of stretch (or strain) on the activation of ONH-derived, ECM-producing cells.