Although it is often recommended to standardize the time of day when performing non-invasive measurements of vascular function, the exact influence of the time of day on the outcome of IMT (intima-media thickness), PWV (pulse wave velocity), AIX (augmentation index) and FMD (flow-mediated dilatation) measurements has not been reported before. Nineteen healthy volunteers visited our department on two different occasions: the first visit was at 09:00 hours after an overnight fast, and the second visit was at 14:00 hours after a standardized breakfast. Non-invasive measurements of atherosclerosis were performed twice at 09:00 hours and once on the second visit at 14:00 hours. Measurement of IMT, PWV, AIX and FMD was reproducible according to the method of Bland and Altman. The absolute difference between repeated measurements at 09:00 hours showed no significant difference compared with the absolute difference between 09:00 and 14:00 hours for IMT (0.029+/-0.014 compared with 0.021+/-0.014 mm; P = 0.27), PWV (0.63+/-0.50 compared with 0.75+/-0.74 m/s; P = 0.52), AIX (4.0+/-4.0 compared with 5.5+/-5.2%; P = 0.35) and FMD (3.8+/-3.7 compared with 4.2+/-2.9%; P = 0.70). In conclusion, our results show that, in healthy volunteers during the daytime, IMT, PWV, AIX and FMD outcomes are not confounded by variation in the exact time of the examination as long as other (exogenous) conditions, including food intake, smoking and intake of alcohol, are carefully controlled for.