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Our purpose was to investigate the spatial and temporal profile of the paraspinal muscle activation during gait in a group of 13 patients with lumbar instability (LI) in a pre-surgical setting compared to the results with those from both 13 healthy controls (HC) and a sample of 7 patients with failed back surgery syndrome (FBSS), which represents a chronic untreatable condition, in which the spine muscles function is expected to be widely impaired. Spatiotemporal gait parameters, trunk kinematics, and muscle activation were measured through a motion analysis system integrated with a surface EMG device. The bilateral paraspinal muscles (longissimus) at L3-L4, L4-L5, and L5-S1 levels and lumbar iliocostalis muscles were evaluated. Statistical analysis revealed significant differences between groups in the step length, step width, and trunk bending and rotation. As regard the EMG analysis, significant differences were found in the cross-correlation, full-width percentage and center of activation values between groups, for all muscles investigated. Patients with LI, showed preserved trunk movements compared to HC but a series of EMG abnormalities of the spinal muscles, in terms of left-right symmetry, top-down synchronization, and spatiotemporal activation and modulation compared to the HC group. In patients with LI some of such EMG abnormalities regarded mainly the segment involved by the instability and were strictly correlated to the pain perception. Conversely, in patients with FBSS the EMG abnormalities regarded all the spinal muscles, irrespective to the segment involved, and were correlated to the disease’s severity. Furthermore, patients with FBSS showed reduced lateral bending and rotation of the trunk and a reduced gait performance and balance. Our methodological approach to analyze the functional status of patients with LI due to spine disease with surgical indications, even in more complex conditions such as deformities, could allow to evaluate the biomechanics of the spine in the preoperative conditions and, in the future, to verify whether and which surgical procedure may either preserve or improve the spine muscle function during gait.