This cross-over study aimed to explore effects of acute whole-body vibration (WBV) at frequencies of 5–35 Hz on heart rate variability and brain excitability. Thirteen healthy physically active college students randomly completed eight interventions under the following conditions: static upright standing without vibration (CON), static squat exercise (knee flexion 150°) on the vibration platform (SSE), and static squat exercise (knee flexion 150°) combined with WBV at vibration frequency of 5, 9, 13, 20, 25, and 35 Hz. Five bouts × 30 s with a 30-s rest interval were performed for all interventions. The brain’s direct current potentials (DCPs), frequency domain variables (FDV) including normalized low frequency power (nLF), normalized high frequency power (nHF) and the ratio of LF to HF (LF/HF), along with the mean heart rate (MHR) were collected and calculated before and after the WBV intervention. Results suggested that WBV frequency at 5 Hz had a substantial effect on decreasing DCPs [−2.13 μV, t(84) = −3.82, p < 0.05, g = −1.03, large] and nLF [−13%, t(84) = −2.31, p = 0.04, g = −0.62, medium]. By contrast, 20–35 Hz of acute WBV intervention considerably improved DCPs [7.58 μV, t(84) = 4.31, p < 0.05, g = 1.16, large], nLF [17%, t(84) = 2.92, p < 0.05, g = 0.79, large] and the LF/HF [0.51, t(84) = 2.86, p < 0.05, g = 0.77, large]. A strong (r = 0.7, p < 0.01) correlation between DCPs and nLF was found at 5 Hz. In summary, acute WBV at 20–35 Hz principally activated the sympathetic nervous system and increased brain excitability, while 5-Hz WBV activated the parasympathetic nervous system and reduced brain excitability. The frequency spectrum of WBV might be manipulated according to the intervention target on heart rate variability and brain excitability.