Shear stresses are powerful regulators of cellular function and potent mediators of the development of vascular disease. We have designed and optimized a system allowing the application of flow to cultured cells in a multichannel format. By using a multichannel peristaltic pump, flow can be driven continuously in the system for long-term studies in multiple isolated flow loops. A key component of the system is a dual-chamber pulse dampener that removes the pulsatility of the flow without the need for having an open system or elevated reservoir. We optimized the design parameters of the pulse dampening chambers for the maximum reduction in flow pulsation while minimizing the fluid needed for each isolated flow channel. Human umbilical vein endothelial cells (HUVECs) were exposed to steady and pulsatile shear stress using the system. We found that cells under steady flow had a marked increased production of eNOS and formation of actin stress fibers in comparison to those under pulsatile flow conditions. Overall, the results confirm the utility of the device as a practical means to apply shear stress to cultured cells in the multichannel format and provide steady, long term flow to microfluidic devices.