This study investigated the dependence of an increasing/decreasing sequence of pulse repetition frequencies (PRFs) on the chronotropic effect via the application of 3.5-MHz pulsed ultrasound (US) on the rat heart. The experiments were divided into three 3-month-old female rat groups (n = 4 ea): control, PRF increase and PRF decrease. Rats were exposed to transthoracic ultrasonic pulses at ~0.50% of duty factor at 2.0-MPa peak rarefactional pressure amplitude. For the PRF increase group, the PRF started lower than that of the rat’s heart rate and was increased sequentially in 1-Hz steps every 5 s (i.e., 4, 5, and 6 Hz) for a total duration of 15 s. For the PRF decrease group, the PRF started greater than that of the rat’s heart rate and was decreased sequentially in 1-Hz steps every 5 s (i.e., 6, 5, and 4 Hz). For the PRF decrease and control groups, the ultrasound application resulted in a significant negative chronotropic effect (~11%) after ultrasound exposure. However, for the PRF increase group, a significant but less decrease of the heart rate (~3%) was observed after ultrasound exposure. The ultrasound application caused a negative chronotropic effect after US exposure for increase/decrease US group.