This study investigated the cumulative effect of a 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. Two 3-mo-old female rat groups (n = 4 ea) were studied: control (US off) and PRF decrease. Rats were exposed to transthoracic ultrasonic pulses at ~0.25% duty factor at 2.0-MPa peak rarefactional pressure amplitude. Three PRF sequences were applied. The PRF started greater than that of the rat’s heart rate and was decreased sequentially in 1-Hz steps every 10 s (i.e., 6, 5 and 4 Hz—one sequence) for a total duration of 30 s. The total US exposure was 90 s (3-sequences). For the PRF decrease group, the ultrasound application resulted in an ~8% significant negative chronotropic effect after the first PRF sequence, ~9% significant negative chronotropic effect after the second PRF sequence, and ~10% significant negative chronotropic effect after the third PRF sequence. No significant changes were observed for the control group. The ultrasound application caused a negative chronotropic effect after US exposure for the PRF decrease group and a slight cumulative effect was observed.