Repetitive transcranial magnetic stimulation (rTMS) system is an important therapeutic tool used in non-invasive brain stimulation. The electric field induced by the time-varying magnetic field in a stimulating coil could activate nerve fibers in the brain, resulting in depolarization or hyperpolarization of the neurons. However, the potential adverse effects of rTMS on heart rhythm have not been extensively investigated. This study aims to develop an optimized design of rTMS system to evaluate the potential adverse effects of rTMS on mouse heart rhythm via vagus nerve modulation for pre-clinical application. The rTMS-induced electric field in the vagus nerve of brain produced by the strong rate of current change of 1.04 × 10 8 A/s in a stimulating coil, which was directly determined by circuit design in charging voltage of the capacitor bank and inductance value of a stimulating coil. A finite element method (FEM) mathematical simulation indicated that the maximum eddy current was 25.4 μA/mm 2 , which was greatly exceeded the vagus nerve activation threshold of 5.6 μA/mm 2 . The animal experiment results also verify that the induced electric field activates the RR-interval prolonging effect might be attributed to vagus nerve stimulation (VNS) from rTMS, and the most pronounced heart rhythm prolonging effect at 20 Hz magnetic field treatment, causing the average heart rate decreased to 58.65% of that before rTMS in 10 mice. In conclusion, above-threshold rTMS at 20 Hz could produce maximum adverse effect on heart rhythm through direct vagus nerve activation for pre-clinical applications such as safety screening.