Whole body vibration has been postulated to contribute to the onset of back pain. However, little is known about the relationship between vibration exposure, the biomechanical response, and the physiological responses of the seated human. The aim of this study was to measure the frequency and corresponding muscle responses of seated male volunteers during whole body vibration exposures along the vertical and anteroposterior directions to define the transmissibility and associated muscle activation responses for relevant whole body vibration exposures. Seated human male volunteers underwent separate whole body vibration exposures in the vertical (Z-direction) and anteroposterior (X-direction) directions using sinusoidal sweeps ranging from 2 to 18 Hz, with a constant amplitude of 0.4 g. For each vibration exposure, the accelerations and displacements of the seat and lumbar and thoracic spines were recorded. In addition, muscle activity in the lumbar and thoracic spines was recorded using electromyography (EMG) and surface electrodes in the lumbar and thoracic region. Transmissibility was determined, and peak transmissibility, displacement, and muscle activity were compared in each of the lumbar and thoracic regions. The peak transmissibility for vertical vibrations occurred at 4 Hz for both the lumbar (1.55 ± 0.34) and thoracic (1.49 ± 0.21) regions. For X-directed seat vibrations, the transmissibility ratio in both spinal regions was highest at 2 Hz but never exceeded a value of 1. The peak muscle response in both spinal regions occurred at frequencies corresponding to the peak transmissibility, regardless of the direction of imposed seat vibration: 4 Hz for the Z-direction and 2–3 Hz for the X-direction. In both vibration directions, spinal displacements occurred primarily in the direction of seat vibration, with little off-axis motion. The occurrence of peak muscle responses at frequencies of peak transmissibility suggests that such frequencies may induce greater muscle activity, leading to muscle fatigue, which could be a contributing mechanism of back pain.