Veterinary professionals are divided on whether to replace needles after drawing up a vaccine, citing concerns about patient discomfort. A recent in vitro study did not find a clinically meaningful difference in the force required to penetrate a standardized material between new needles and those that had passed once or twice through a vial septum.1 However, no in vivo studies have evaluated whether needle replacement affects dogs clinically. This study aimed to determine whether needle replacement reduces behavioral or physiological signs of discomfort in dogs and whether vaccine administrators can subjectively detect needle replacement.