The pathogenesis of lumbar adjacent segment disease is thought to be secondary to altered biomechanics resulting from fusion. Direct in vivo evidence for altered biomechanics following lumbar fusion is lacking. This study’s aim was to describe in vivo kinematics of the superior adjacent segment relative to the fused segment before and after lumbar fusion. This study analyzed seven patients with symptomatic lumbar degenerative spondylolisthesis (5 M, 2F; age 65 ± 5.1 years) using a biplane radiographic imaging system. Each subject performed two to three trials of continuous flexion of their torso according to established protocols. Synchronized biplane radiographs were acquired at 20 images per second one month before and six months after single-level fusion at L4-L5 or L5-S1, or two-level fusion at L3-L5 or L4-S1. A previously validated volumetric model-based tracking process was used to track the position and orientation of vertebrae in the radiographic images. Intervertebral flexion/extension and AP translation (slip) at the superior adjacent segment were calculated over the entire dynamic flexion activity. Skin-mounted surface markers were tracked using conventional motion analysis and used to determine torso flexion. Change in adjacent segment kinematics after fusion was determined at corresponding angles of dynamic torso flexion. Changes in adjacent segment motion varied across patients, however, all patients maintained or increased the amount of adjacent segment slip or intervertebral flexion/extension. No patients demonstrated both decreased adjacent segment slip and decreased rotation. This study suggests that short-term changes in kinematics at the superior adjacent segment after lumbar fusion appear to be patient-specific.