It has been well established that wall shear stress is an important determinant of endothelial cell function and gene expression as well as of its structure. There is increasing evidence that low wall shear stress, as pres- ent in artery bifurcations opposite to the flow divider where atherosclerotic lesions preferentially originate, expresses an atherogenic endothelial gene profile. Besides, wall shear stress regulates arterial diameter by modifying the release of vasoactive mediators by endothelial cells. Most of the studies on the influence of wall shear stress on endothelial cell function and structure have been performed in vitro, generally exposing endothelial cells from different vascular regions to an average wall shear stress level calculated according to Poiseuille’s law, which does not hold for the in vivo situation, assuming wall shear stress to be constant along the arterial tree. Also in vivo wall shear stress has been determined based upon theory, assuming the velocity profile in arteries to be parabolic, which is generally not the case. Wall shear stress has been calculated, because of the lack of techniques to assess wall shear stress in vivo. In recent years, techniques have been developed to accurately assess velocity profiles in arterioles, using fluorescently labeled particles as flow tracers, and non-invasively in large arteries by means of ultrasound or magnetic resonance imaging. Wall shear rate is derived from the in vivo recorded velocity profiles and wall shear stress is estimated as the product of wall shear rate and plasma viscosity in arterioles and whole blood viscosity in large arteries. In this review, we will discuss wall shear stress in vivo, paying attention to its assessment and especially to the results obtained in both arterioles and large arteries. The limitations of the methods currently in use are discussed as well. The data obtained in the arterial system in vivo are compared with the theoretically predicted ones, and the consequences of values deviating from theory for in vitro studies are considered. Applications of wall shear stress as in flow-mediated arterial dilation, clinically in use to assess endothelial cell (dys)function, are also addressed. This review starts with some background considerations and some theoretical aspects.