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This study investigated the influence of five different muscle groups on the monosegmental motion (L4–L5) during pure flexion/extension, lateral bending, and axial rotation moments.The results showed and compared the effect of different muscle groups acting in different directions on the stability of a single motion segment to find loading conditions of in vitro experiments that simulate more physiologically reasonable loads.In spine biomechanics research, most in vitro experiments have been carried out without applying muscle forces, even though these forces stabilize the spinal column in vivo.Seven human lumbosacral spines were tested in a spine tester that allows simulation of up to five symmetrical muscle forces. Changing pure flexion/extention, lateral binding, and axial rotation moments up to ±3.75 Nm were applied without muscle forces, with different muscle groups and combinations. The three-dimensional monosegmental motion was determined using an instrumented spatial linkage system.Simulated muscle forces were found to strongly influence load-deformation characteristics. Muscle action generally increased the range of motion and the natural zone of the motion segments. This was most evident for flexion and extension. After five pairs of symmetrical, constant muscle forces were applied (80 N per pair) the range of motion decreased about 93% in flexion and 85% in extension. The total natural zone for flexion and extension was decreased by 83% muscle action. The multifluids muscle group had the strongest influence.This experiment showed the important of including at least some of the most important muscle groups in invitro experiments in lumbar spine specimens.