Atherosclerotic lesions occur non-randomly at vascular niches in bends and bifurcations where fluid flow can be characterized as "disturbed" (low shear stress with both forward and retrograde flow). Endothelial cells (ECs) at these locations experience significantly lower average shear stress without change in the levels of pressure or strain, which affects the local balance in mechanical stresses. Common in vitro models of atherosclerosis focus primarily on shear stress without accounting for pressure and strain loading. To overcome this limitation, we used our microfluidic endothelial cell culture model (ECCM) to achieve accurate replication of pressure, strain, and shear stress waveforms associated with both normal flow seen in straight sections of arteries and disturbed flow seen in the abdominal aorta in the infrarenal segment at the wall distal to the inferior mesenteric artery (IMA), which is associated with high incidence of atherosclerotic lesion formation. Human aortic endothelial cells (HAECs) were cultured within the ECCM under both normal and disturbed flow and evaluated for cell shape, cytoskeletal alignment, endothelial barrier function, and inflammation using immunofluorescence microscopy and flow cytometry. Results clearly demonstrate quantifiable differences between cells cultured under disturbed flow conditions, which are cuboidal with short and randomly oriented actin microfilaments and show intermittent expression of β-Catenin and cells cultured under normal flow. However, in the absence of pro-inflammatory stimulation, the levels of expression of activation markers: intra cellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), and vascular endothelial cell growth factor - receptor 2 (VEGF-R2) known to be involved in the initiation of plaque formation were only slightly higher in HAECs cultured under disturbed flow in comparison to cells cultured under normal flow. © 2011 American Institute of Physics.