High resolution in vivo velocity measurements within the cardiovascular system are essential for accurate calculation of vessel wall shear stress, a highly influential factor for the progression of arterial disease. Unfortunately, currently available techniques for in vivo imaging are unable to provide the temporal resolution required for velocity measurement at physiological flow rates. Advances in technology and improvements in imaging systems are allowing a relatively new technique, X-ray velocimetry, to become a viable tool for such measurements. This study investigates the haemodynamics of pulsatile blood flow in an optically opaque in vitro model at physiological flow rates using X-ray velocimetry. The in vitro model, an asymmetric stenosis, is designed as a 3:1 femoral artery with the diameter and flow rate replicating vasculature of a mouse. Velocity measurements are obtained over multiple cycles of the periodic flow at high temporal and spatial resolution (1 ms and 29 μm, respectively) allowing accurate measurement of the velocity gradients and calculation of the wall shear stress. This study clearly illustrates the capability of in vitro X-ray velocimetry, suggesting it as a possible measurement technique for future in vivo vascular wall shear stress measurement.