The Valsalva maneuver is known to affect intraocular pressure (IOP). Simple breath-holding may cause IOP elevation. A recent study demonstrated a decrease in pulsatile ocular blood flow (POBF) during forcible exhalation. This study investigated whether breath-holding could affect POBF measurement. Thirty-eight healthy young adults were recruited. Their POBF's were measured with an ocular blood flow pneumatonometer. The first set of measurements was made using normal measurement protocol. Three consecutive readings were obtained, and the mean was used for analysis. The second set of measurements was taken after 5-min rest, and the subjects were required to hold their breath during the acquisition period. As previously reported, POBF reduced with increasing myopia. There was no significant change in IOP, pulse rate, POBF, and pulse amplitude between normal protocol and breath-holding condition. POBF and pulse amplitude demonstrated a greater variation, shown by coefficient of variation, when subjects held their breath. This study found a greater variation in consecutive POBF measurements during breath-holding condition. There was no significant difference in either POBF or pulse amplitude during breath-holding session, probably because of the use of three consecutive measurements, and averaged results were generated from them. It is advised to measure the POBF by taking consecutive readings, and subjects should not hold their breath.