Habitual short sleep durations are associated with several cardiovascular diseases. Experimental research generally supports these findings as metrics of arterial function are impaired after complete deprivation of sleep and after longer periods of partial sleep restriction. The acute influence of a single instance of partial sleep restriction (PSR), however, has not been defined. We evaluated arterial structure and function among 32 university-aged participants on two occasions: once after normal habitual sleep (NS), and again the morning after an acute partial sleep restriction (PSR) intervention involving only 3h of sleep for a single night. Endothelial function was measured using ultrasonography at the brachial artery via flow-mediated dilatation (FMD), and a ramp peak oxygen uptake test was used to evaluate cardiorespiratory fitness. Blood samples were collected from a subset of participants to investigate the influence of circulatory factors on cellular mechanisms implicated in endothelial function. Sleep duration was lower after a night of PSR compared to NS (P<0.001); however, there were no appreciable differences in any haemodynamic outcome between conditions. FMD was not different between NS and PSR (NS: 6.5±2.9%; PSR: 6.3±2.9%; P=0.668), and cardiorespiratory fitness did not moderate the haemodynamic response to PSR (all P>0.05). Ex vivo cell culture results aligned with in vivo data, showing that acute PSR does not alter intracellular processes involved in endothelial function. No differences in arterial structure or function were observed between NS and acute PSR in healthy and young participants, and cardiorespiratory fitness does not modulate the arterial response to acute sleep restriction.