Wall shear stress (WSS) is an important stimulus in vascular remodelling and vascular lesion development. The current methods to assess and predict the risk associated with specific unsteady WSS consider the WSS mean values or the presence of reverse phases described by the oscillatory shear index. Recent evidence has shown that the accuracy of these methods is limited, especially with respect to the venous environment. Unsteady WSS are characterised by several features that may individually affect endothelial cells. Consequently, we assessed the effects of averaged WSS (TAWSS), temporal WSS gradient (TWSSG), maximum WSS (WSS peak) and reverse phase (OSI) by applying different WSS profiles to venous EC in-vitro, using a real-time controlled cone-and-plate cell-shearing device for 24 h. We found that TWSSG and WSS peak affect cell elongation and alignment respectively. We also found that the WSS waveforms with a peak of 1.5 Pa or higher significantly correlate with the induction of a protective phenotype. Cell phenotype induced by these high peak waveforms does not correlate to what is predicted by the hemodynamic indices currently used. The definition of reliable hemodynamic indices can be used to inform the computational models aimed at estimating the hemodynamic effects on vascular remodelling.