Human endothelial cells (hECs) experience complex spatiotemporal hemodynamic flows and that directly regulate hEC function and susceptibility to cardiovascular disease. Recent medical imaging studies reveal that helical flows strongly correlate with lowered disease susceptibility, as contrasted to multidirectional disturbed flows. However, a lack of platforms to replicate these spatial profiles of flow (SPF) has prevented biological studies to investigate the role hECs play in tuning the observed SPF-correlated disease susceptibility. Here, we utilize microfluidic devices to apply varying SPF upon hECs for the first time, and discover that these flows can differentially impact hEC morphology, transcription, and polarization. Collectively, our platform and studies significantly advance our ability to delineate flow-regulated hEC function and disease susceptibility.Significance StatementIn vivo, hECs experience complex hemodynamic flows, including those that are spatially helical or disturbed, which is in stark contrast to the unidirectional flows typically used to study hECs in vitro. Understanding the impact of SPF on hEC function informs our understanding of the pathophysiology of hEC dysfunction and can lead to interventional solutions that specifically perturb SPF to lower disease risk. Here, we leverage microfluidics to apply and discover the specific impact of SPF on hECs for the first time. Broadly, our platform bridges the mutual interests of the vascular biology and interventional cardiology communities to collectively understand how cardiovascular health is tied to the way blood flows upon the endothelium.