There is a strong time-of-day variation in vasoconstriction in response to sympathetic stimulation that may contribute to the time-of-day variation in blood pressure, which is characterised by a dip in blood pressure during the resting-period when sympathetic activity is low. Vasoconstriction is known to be tightly regulated by nitric-oxide signalling from the endothelial cells, so we have looked at the effect of the time-of-day on the ability of endothelial nitric oxide synthase (eNOS) signalling to modulate of vascular contractility. We used wire-myography to measure contractile force from 2-4 mm ring segments of the superior mesenteric artery 1st order branches isolated at two opposing time points corresponding to the nocturnal Wistar rats active-period (night) and resting-period (day). Our data showed that mesenteric resistance vessels exhibit a time-of-day variation in their contractile-response to α1-adrenoreceptor (ADR) and muscarinic activation, characterised by a reduced vasoconstriction in response to a concentration of phenylephrine and enhanced vasodilation in response to acetylcholine during the active- versus the resting-period. This reduced vasoconstriction in response to phenylephrine is also present in response to high-K contractions, suggesting a mechanism independent of the signalling pathway involved in α1-ADR activation. This time-of-day difference in response to phenylephrine and acetylcholine is absent in the presence of L-Nitro-Arginine Methyl Ester (L-NAME), and the variation in contraction in response to high-K requires an intact endothelium. We found a large increase in eNOS expression (mRNA and protein) during the active- versus resting-period vessels, which may reflect the presence of a functioning peripheral circadian clock in mesenteric resistance vessels. The peak in eNOS-signalling during the active-period and its dampening effect on vasoconstriction in response to α1-ADR activation would reduce any sympathetic-driven rise in peripheral resistance, which combined with the reduction in the positive inotropic response of the ventricles to sympathetic stimulation (Collins & Rodrigo, 2010), would limit the rise in blood pressure with a beneficial impact on stroke and hypertensive heart disease. This time-of-day variation in endothelium-derived nitric oxide signalling may also have far reaching physiological consequences over and above regulation of blood pressure. For example; elevated nitrite levels in the blood have been linked to cardioprotection from remote ischaemic conditioning and nitric oxide in response to eNOS activation by sheer stress has an anti-platelet aggregation action. Thus an increase in eNOS activity during the active period may play a protective role against cardiovascular pathologies.