Deep brain stimulation has been used to treat movement disorders such as Parkinson diseases by utilizing high-frequency stimulations (HFS) of electrical pulses with constant inter-pulse-intervals (IPI). To develop new stimulation paradigms for treating more brain diseases, HFS with varying IPI (i.e., varying-frequency) has been investigated. Previous studies have shown that the efficacy obtained by varying-frequency is different from that obtained by constant-frequency even with a same mean frequency. We hypothesized that small changes in IPI during HFS could substantially change the effect of HFS on neurons. To test this hypothesis, HFS sequences with constant IPI (IPI = 10 or 7.5 ms for a frequency of 100 or 133 Hz ) and varying IPI (IPI = 5-10 ms with a mean frequency of 133 Hz) were alternately applied at afferent axon fibers of pyramidal cells in rat hippocampal CA1 region. The evoked potentials of downstream neurons were recorded and analyzed to quantitatively evaluate the neuronal responses to stimulations with constant IPI and varying IPI. The results showed that during persistent stimulation with constant IPI, the responses of downstream neurons changed from initial synchronized firing of population spikes (PS) into non-synchronized firing (i.e., unit spikes). However, once the stimulation switched to the sequence with varying IPI, synchronized firing reappeared with large PS events. Additionally, the amplitude of PS and the synchronization degree of firing induced by varying IPI were similar to those induced by single pulses at baseline. However, the incidence of PS was only ~7% of the pulse frequency, indicating a cumulative action of multiple pulses for generating such synchronized firing of neurons by stimulations with varying IPI. In addition, the appearance of PS was related to the length of proceeding IPI. Presumably, nonlinear responses of neuronal axons and synapses to high-frequency stimulation might cause the synchronized activity induced by varying-frequency. These results indicate that tiny differences in intervals of varying-frequency stimulation may generate a modulation effect on neurons very different from that of constant-frequency stimulation. The present study shows important results for revealing the mechanisms of brain stimulation and for advancing the development of new stimulation paradigms to treat various brain diseases.