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We introduce a microfluidic impedance platform to electrically monitor in real-time, endothelium monolayers undergoing fluid shear stress. Our platform incorporates sensing electrodes (SEs) that measure cell behavior and cell-free control electrodes that measure cell culture media resistance simultaneously but independently from SEs. We evaluated three different cellular subpopulations sizes through 50, 100, and 200 μm diameter SEs. We tested their utility in measuring the response of human umbilical vein endothelial cells (HUVECs) at static, constant (17.6 dyne per cm2), and stepped (23.7–35–58.1 dyne per cm2) shear stress conditions. For 14 hours, we collected the impedance spectra (100 Hz–1 MHz) of sheared cells. Using equivalent circuit models, we extracted monolayer permeability (RTER), cell membrane capacitance, and cell culture media resistance. Platform evaluation concluded that: (1) 50 μm …