The human spinal column, devoid of musculature, is incapable of carrying normal physiologic loads. In an in vitro experiment, the effect of simulated intersegmental muscle forces on spinal instability was investigated. Intact and sequentially injured fresh lumbar functional spinal units were subjected to three-dimensional biomechanical tests with increasing muscle forces. With the application of muscle forces, range of motion (ROM) increased and neutral zone (NZ) decreased in flexion loading, while both ROM and NZ decreased in extension loading. In lateral bending, ROM and NZ were unaffected by the application of the muscle forces. In axial rotation, ROM decreased significantly, while NZ decrease was statistically insignificant. It was concluded that the action of the intersegmental muscle forces is to maintain or decrease intervertebral motions after injury, with the exception of the flexion ROM, which increased with the application of muscle forces. In addition, the study suggested that Neutral Zone is a better indicator of spinal instability than Range of Motion.