Adjacent-level disease is a common iatrogenic complication seen among patients undergoing spinal fusion for low back pain. This is attributed to the postsurgical differences in stiffness between the spinal levels, which result in abnormal forces, stress shielding, and hypermobility at the adjacent levels. In addition, as most patients undergoing these surgeries are osteoporotic, screw loosening at the index level is a complication that commonly accompanies adjacent-level disease. Recent studies indicate that a rod with lower rigidity than that of titanium may help to overcome these detrimental effects at the adjacent level. The present study was conducted in vitro using 12 L1-S1 specimens divided into groups of six, with each group instrumented with either titanium rods or PEEK (polyetheretherketone) rods. The test protocol included subjecting intact specimens to pure moments of 10 Nm in extension and flexion using an FS20 Biomechanical Spine Test System (Applied Test Systems) followed by hybrid moments on the instrumented specimens to achieve the same L1-S1 motion as that of the intact specimens. During the protocol's later phase, the L4-L5 units from each specimen were segmented for cyclic loading followed by postfatigue kinematic analysis to highlight the differences in motion pre- and postfatigue. The objectives included the in vitro comparison of (1) the adjacent-level motion before and after instrumentation with PEEK and titanium rods and (2) the pre- and postfatigue motion at the instrumented level with PEEK and titanium rods. The results showed that the adjacent levels above the instrumentation caused increased flexion and extension with both PEEK and titanium rods. The postfatigue kinematic data showed that the motion at the instrumented level (L4-L5) increased significantly in both flexion and extension compared to prefatigue motion in titanium groups. However, there was no significant difference in motion between the pre- and postfatigue data in the PEEK group.