When rat pups are isolated from their mothers, they emit ultrasonic vocalizations (USVs). Although previous studies have reported that USVs are related to anxiety, others have reported that they are related to simple, nonemotional factors, such as physiological reactions to coldness. In this study, we examined the influence of three maternal separations on rat pups. The number of USVs during 5 min of USV test under maternal separation, latency in the righting reflex as motor function, and body temperature were recorded twice (the first and second tests) before and after the pups were put in various environments for 10 min. The environments were no maternal separation (Control: CON), maternal separation with littermates (LMS), and single maternal separation with a heater (SMS). In the second test, the SMS pups had fewer USVs, a lower body temperature, and a more rapid righting reflex than the CON and LMS pups. In addition, there was no strong correlation between USVs and righting reflex. As a result, pups undergoing 10 min of SMS while being kept warm by the heater showed rapid righting reflex. Thus, by a single maternal separation, the number of USVs decreased but the decrease was unrelated to decrease in motor function.