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Disturbed fluid flow is well understood to have significant ramifications on endothelial function, but the impact disturbed flow has on endothelial biomechanics is not well understood. In this study, we measured tractions, intercellular stresses, and cell velocity of endothelial cells exposed to disturbed flow using a custom-fabricated f low chamber. Our flow chamber exposed cells to disturbed fluid flow within the following spatial zones: zone 1 (inlet; length 0.676–2.027 cm): 0.0037 ± 0.0001 Pa; zone 2 (middle; length 2.027–3.716 cm): 0.0059 ± 0.0005 Pa; and zone 3 (outlet; length 3.716–5.405 cm): 0.0051 ± 0.0025 Pa. Tractions and intercellular stresses were observed to be highest in the middle of the chamber (zone 2) and lowest at the chamber outlet (zone 3), while cell velocity was highest near the chamber inlet (zone 1), and lowest near the middle of the chamber (zone 2). Our findings suggest endothelial biomechanical response to disturbed fluid flow to be dependent on not only shear stress magnitude, but the spatial shear stress gradient as well. We believe our results will be useful to a host of fields including endothelial cell biology, the cardiovascular field, and cellular biomechanics in general.