Peristalsis is a nuanced mechanical stimulus comprised of multi-axial strain (radial and axial strain) and shear stress. Forces associated with peristalsis regulate diverse biological functions including digestion, reproductive function, and urine dynamics. Given the central role peristalsis plays in physiology and pathophysiology, we were motivated to design a bioreactor capable of holistically mimicking peristalsis. We engineered a novel rotating screw-drive based design combined with a peristaltic pump, in order to deliver multiaxial strain and concurrent shear stress to a biocompatible polydimethylsiloxane (PDMS) membrane “wall”. Radial indentation and rotation of the screw drive against the wall demonstrated multi-axial strain evaluated via finite element modeling. Experimental measurements of strain using piezoelectric strain resistors were in close alignment of model-predicted values (15.9 ± 4.2% vs. 15.2% predicted). Modeling of shear stress on the ‘wall’ indicated a uniform velocity profile and a moderate shear stress of 0.4 Pa. Human mesenchymal stem cells (hMSCs) seeded on the PDMS ‘wall’ and stimulated with peristalsis demonstrated dramatic changes in actin filament alignment, proliferation, and nuclear morphology compared to static controls, perfusion or strain, indicating that hMSCs sensed and responded to peristalsis uniquely. Lastly, significant differences were observed in gene expression patterns of Calponin, Caldesmon, Smooth Muscle Actin, and Transgelin, corroborating the propensity of hMSCs toward myogenic differentiation in response to peristalsis. Collectively, our data suggests that the peristalsis bioreactor is capable of generating concurrent multi-axial strain and shear stress on a ‘wall’. hMSCs experience peristalsis differently than perfusion or strain, resulting in changes in proliferation, actin fiber organization, smooth muscle actin expression, and genetic markers of differentiation. The peristalsis bioreactor device has broad utility in the study of development and disease in several organ systems.