Study Design: An in vitro study on human multilevel spinal segments. Objective: To determine the differences in biomechanical characteristics between 4 separate regions of the human spine and to provide quantitative information is derived on the range of motion (ROM), neutral zone (NZ), neutral zone stiffness (NZstiff), and flexibility (FLEX). Summary of Background Data: Limited literature is available about the biomechanical behavior of different regions of the human spine, in particular with multilevel segments. Test setup en protocols were different between studies and therefore outcomes of separate regions are hardly comparable. Methods: A total of 24 spinal segments of 6 human cadaveric spines were prepared for biomechanical testing. Each specimen contained 4 vertebrae and 3 intervertebral discs: T1-T4, T5-T8, T9-T12, and L1-L4. Pure moments were applied to a maximum of 4 Nm in flexion/extension, lateral bending, and axial rotation. Displacement of individual motion segments was measured using a 3-dimensional movement registration system. ROM, NZ, NZstiff, and FLEX of the spinal regions were calculated from the acquired load-displacement data. Results: In axial direction, ROM and NZ decreased and NZ stiffness increased from high to low vertebral levels. For flexion/extension and lateral flexion highest ROM and NZ and lowest NZ stiffness values were found at the T1-T4 and L1-L4 regions. NZ magnitudes and NZ stiffnesses were negatively correlated (P < 0.05). Flexibility of the spinal regions was variable; no significant differences were found between the 4 spinal regions. Conclusion: This study showed the differences in ROM, NZ, and NZ stiffness between thoracolumbar regions of the human spine in axial rotation, flexion/extension, and lateral bending. Separate multilevel spinal segments were tested in 1 study, and therefore characteristics of different regions are truly comparable. © 2009, Lippincott Williams & Wilkins.