Objective To study the stress on elastic substrate of an in vitro endothelial cell dynamic culture device, whose hemodynamic environment is designed to simulate the human body, and to test and observe the shear stress changes in elastic substrate of parallel plate flow chamber under different tensile stresses. Methods A series of static tensile tests were adopted to fit the condition of dynamic stretching. Namely, the silicone sheet with 2 different thicknesses were put into the device, and then applied with static stretch at the interval of 10% tensile rate (0%, 10%, 20%, 30%), and under the condition of maintaining its tensile rate, the chamber height after the stretch of silicon sheet was calculated. Based on the calculation of shear stress, shear stress curves at different tensile rates were obtained, to make comparative analysis on variation of the shear stress with the thickness of silicon sheet. Results The experimental result was consistent with the theoretical analysis. When the tensile rate was 30%, silicon sheet with 0.5 mm thickness would produce certain influence on shear stress of parallel plate flow chamber along with the change of tensile rate (the height of chamber), and the average and maximum shear stress were reduced by 10.1% and 10.4%, respectively. Conclusions The influence factors caused by the change of elastic substrate thickness after the introduction of tensile stress must be taken into account for the calculation of shear stress in parallel plate flow chamber. The result can provide experimental technology for the culture of endothelial cells in vitro and the design and development of novel parallel plate flow chamber.