Background Pulsatile ow has been proved to protect vital organ function and microcirculation during extracorporeal membrane oxygenation (ECMO). Studies revealed that pulsatile shear stress plays a vital role in the microcirculatory function and integrity. The objective of this study was to investigate how pulsatility affects wall shear stress and microcirculation during ECMO. Methods Using the i-Cor system, we compared the effects of pulstile or non-pulsatile ows in a canine ECMO model, with hemodynamic parameters and peak wall shear stress (PWSS) calculated. Serum concentrations of syndecan-1 and heparan sulfate were measured at different time points during ECMO. Pulstile shear stress experiments were also validated in endothelial cells exposed to different magnitude of pulsatility, with cell viability, the expressions of syndecan-1 and endothelial-to-mesenchymal tranformation (EndMT) markers analyzed. Results The pulsatile ow generated more surplus hemodynamic energy and preserved higher PWSS during ECMO. Serum concentrations of syndecan-1 and heparan sulfate were both negatively correlated with PWSS, and signi cantly lower levels were observed in the pulsatile group. In addition, non-pulsatility triggered EndMT, with EndMT related genes up-regulated, and endothelial cells exposed to low pulsatility had the lowest possibility of EndMT. Conclusion The maintenance of the PWSS by pulsatility during ECMO contributes to the bene cial effects on glycocalyx integrity and microcirculatory function. Moreover, pulsatility prevents EndMT in endothelial cells, and low pulsatility exhibits the best protective effects. The augmentation of pulsatility may be a future direction to improve the clinical outcome in ECMO.