When neural tissue is stretched, the neurons can undergo injury and impaired function [1,2]. Nerve fibers and fibroblasts are mechanosensitive [3,4]. Both cell types not only react to direct loading but can also initiate responses when exposed to indirect loading, which can occur when their surrounding extracellular matrix components, like collagen, are distorted [5,6]. As a result of mechanical stimuli, these cells can release chemical factors that regulate neuronal activity [7,8]. Although neuronal function and activity have been studied in the context of macroscopic stretch [8,9], their relationship to local strains and the effects of fibroblasts, which are known to alter the local mechanical environment[10] is less clear. In this study, cortical neurons were seeded on microsphere-embedded collagen gels and exposed to macroscopic gel stretches of different magnitudes with physiologic and injury relevance to test the hypothesis that the presence of fibroblasts in a co-culture system changes local strains and modulates neuronal activity.