Cyclic myometrial contractions of the non-pregnant uterus induce intra-uterine peristaltic flows, which have important roles in transport of sperm and embryos during early stages of reproduction. Hyperperistalsis in young females may lead to migration of endometrial cells and development of adenomyosis or endometriosis. We conducted an in vitro study of the biological response of a tissue engineered endometrial barrier exposed to peristaltic wall shear stresses (PWSSs). The endometrial barrier model was co-cultured of endometrial epithelial cells on top of myometrial smooth muscle cells (MSMCs) in custom-designed wells that can be disassembled for mechanobiology experiments. A new experimental setup was developed for exposing the uterine wall in vitro model to PWSSs that mimic the in vivo intra-uterine environment. Peristaltic flow was induced by moving a belt with bulges to deform the elastic cover of a fluid filled chamber that held the uterine wall model at the bottom. The in vitro biological model was exposed to peristaltic flows for 60 and 120 min and then stained for immunofluorescence studies of alternations in the cytoskeleton. Quantification of the F-actin mass in both layers revealed a significant increase with the length of exposure to PWSSs. Moreover, the inner layer of MSMCs that were not in direct contact with the fluid also responded with an increase in the F-actin mass. This new experimental approach can be expanded to in vitro studies of multiple structural changes and genetic expressions, while the tissue engineered uterine wall models are tested under conditions that mimic the in vivo physiological environment. © 2020 Author(s).