Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and an important contributor to morbidity and mortality. Endothelial dysfunction has been postulated to be an important contributing factor in cardiovascular events in patients with AF. However, how vascular endothelial cells respond to arrhythmic flow is not fully understood, mainly due to the limitation of current in vitro systems to mimic arrhythmic flow conditions. To address this limitation, we developed a microfluidic system to study the effect of arrhythmic flow on the mechanobiology of human aortic endothelial cells (HAECs). The system utilises a computer-controlled piezoelectric pump for generating arrhythmic flow with a unique ability to control the variability in both the frequency and amplitude of pulse waves. The flow rate is modulated to reflect physiological or pathophysiological shear stress levels on endothelial cells. This enabled us to systematically dissect the importance of variability in the frequency and amplitude of pulses and shear stress level on endothelial cell mechanobiology. Our results indicated that arrhythmic flow at physiological shear stress level promotes endothelial cell spreading and reduces the plasma membrane-to-cytoplasmic distribution of β-catenin. In contrast, arrhythmic flow at low and atherogenic shear stress levels does not promote endothelial cell spreading or redistribution of β-catenin. Interestingly, under both shear stress levels, arrhythmic flow induces inflammation by promoting monocyte adhesion via an increase in ICAM-1 expression. Collectively, our microfluidic system provides opportunities to study the effect of arrhythmic flows on vascular endothelial mechanobiology in a systematic and reproducible manner.