Objective To mitigate the risk of iatrogenic instability, new posterior decompression techniques able to preserve musculoskeletal structures have been introduced but never extensively investigated from a biomechanical point of view. This study was aimed to investigate the impact on spinal flexibility caused by a unilateral laminotomy for bilateral decompression, in comparison to the intact condition and a laminectomy with preservation of a bony bridge at the vertebral arch. Secondary aims were to investigate the biomechanical effects of two-level decompression and the quantification of the restoration of stability after posterior fixation. Methods A universal spine tester was used to measure the flexibility of six L2-L5 human spine specimens in intact conditions and after decompression and fixation surgeries. An incremental damage protocol was applied : 1) unilateral laminotomy for bilateral decompression at L3-L4; 2) on three specimens, the unilateral laminotomy was extended to L4-L5; 3) laminectomy with preservation of a bony bridge at the vertebral arch (at L3-L4 in the first three specimens and at L4-L5 in the rest); and 4) pedicle screw fixation at the involved levels. Results Unilateral laminotomy for bilateral decompression had a minor influence on the lumbar flexibility. In flexion-extension, the median range of motion increased by 8%. The bone-preserving laminectomy did not cause major changes in spinal flexibility. Two-level decompression approximately induced a twofold destabilization compared to the single-level treatment, with greater effect on the lower level. Posterior fixation reduced the flexibility to values lower than in the intact conditions in all cases. Conclusion In vitro testing of human lumbar specimens revealed that unilateral laminotomy for bilateral decompression and bone-preserving laminectomy induced a minor destabilization at the operated level. In absence of other pathological factors (e.g., clinical instability, spondylolisthesis), both techniques appear to be safe from a biomechanical point of view.