To investigate whether a nitric oxide donor given as a single oral dose is able to modify aqueous humour flow in healthy volunteers. Ten healthy volunteers participated in a randomized, double-masked and placebo-controlled cross-over study. Aqueous humour flow was assessed by fluorophotometry after intake of isosorbide-5-mononitrate (ISMN), 10 mg. Topical timolol maleate, which is known to reduce aqueous humour flow, was used as a positive control. Intraocular pressure (IOP) was measured by applanation tonometry and blood pressure was registered. The basal rate of aqueous humour flow did not change significantly after a single oral dose of ISMN. The aqueous humour flow in the timolol-treated eye was reduced as compared to the contralateral control eye (p = 0.002). Mean IOP 6 hours after placebo and ISMN intake did not differ significantly. Timolol lowered IOP by 4 mmHg (p < 0.001). ISMN did not lower systolic blood pressure, but diastolic blood pressure was reduced by 4 mmHg (p = 0.048). A single oral dose of 10 mg ISMN had no significant effect on aqueous humour flow in healthy volunteers.