Endothelial cells (ECs) are continuously exposed in vivo to cyclic strain and shear stress from pulsatile blood flow. When these stimuli are applied in vitro, ECs adopt an appearance resembling their in vivo state, most apparent in their alignment (perpendicular to uniaxial strain and along the flow). Uniaxial strain and flow perpendicular to the strain, used in most in vitro studies, only represent the in vivo conditions in straight parts of vessels. The conditions present over large fractions of the vasculature can be better represented by anisotropic biaxial strains at various orientations to flow. To emulate these biological complexities in vitro, we have developed a medium-throughput device to screen for the effects on cells of variously oriented anisotropic biaxial strains and flow combinations. Upon the application of only strains for 24 h, ECs (HUVECs) aligned perpendicular to the maximum principal strain and the alignment was stronger for a higher maximum:minimum principal strain ratio. A 0.55 Pa shear stress, when applied alone or with strain for 24 h, caused cells to align along the flow. Studying EC response to such combined physiological mechanical stimuli was not possible with existing platforms and to our best knowledge, has not been reported before.