(1) To investigate the effect of performing sequential measurements using a Pulsatile Ocular Blood Flow (POBF) Tonograph on POBF and intraocular pressure (IOP) measurements; (2) to determine any effect on IOP caused by performing POBF measurements on the fellow eye; and (3) to assess repeatability of measurements obtained using the POBF Tonograph. Forty-six normal adult subjects (13 men, 33 women; age range, 18 to 55 years) took part in the present study. Twenty-one subjects underwent sequential measurements with a POBF Tonograph on five different occasions with five different time intervals (1, 2, 5, 10, and 15 minutes) between recordings. Twenty subjects underwent a baseline measurement on one eye with the POBF Tonograph, followed by repeat measurements in both eyes after 15 minutes. An additional 25 subjects underwent sequential measurements on the same eye with an interval of 15 minutes between readings. The intraclass correlation coefficient and Bland-Altman plots were used to assess repeatability of the instrument for all 46 subjects for the 15-minute time interval. In all the groups, repeated measurement resulted in a lower recorded IOP. A multivariate analysis of variance indicated that there was a significant decrease in IOP when measurements were repeated within 15 minutes (p=0.024), but there was no significant change in the POBF values (p=0.76). The recorded IOP was significantly lower when sequential measurements were performed with an interval of 1 minute (p <0.01) compared with longer time intervals. The decrease in IOP between baseline and the repeat measurement was significantly greater in the 1-minute interval group compared with the 15-minute interval group (p <0.005). There was no significant difference in POBF values between the baseline and repeat measurements in any group. There was no significant change in IOP induced by a single measurement on the fellow eye (p >0.05). The intraclass correlation coefficient value indicated good agreement between the values of the baseline and repeat measurements using the POBF Tonograph but had wide upper and lower limits of agreement. If IOP measurements have to be repeated using the POBF Tonograph, they are best done after an interval of at least 2 minutes and preferably after 15 minutes. Use of the POBF Tonograph had no significant immediate effect on the IOP or POBF values obtained from a fellow eye.