The development of adjacent segment degeneration (ASD) is a major concern after lumbar spinal fusion surgery, but the causative mechanisms remain unclear. This study used a combined in vivo and in silico method to investigate the changes of anatomical dimensions and biomechanical responses of the adjacent segment (L3-4) after spinal fusion (L4-S1) in five patients under weight-bearing upright standing conditions. The in vivo adjacent disc height changes before and after fusion were measured using a dual fluoroscopic imaging system (DFIS), and the measured in vivo intervertebral positions and orientations were used as displacement boundary conditions of the patient-specific three-dimensional (3D) finite element (FE) disc models to simulate the biomechanical responses of adjacent discs to fusion of the diseased segments. Our data (represented by medians and 95% confidence intervals) showed that a significant decrease by – 0.8 (- 1.2, – 0.4) mm (p < 0.05) in the adjacent disc heights occurred at the posterior region after fusion. The significant increases in disc tissue strains and stresses, 0.32 (0.21, 0.43) mm/mm (p < 0.05) and 1.70 (1.07, 3.60) MPa (p < 0.05), respectively, after fusion were found in the posterolateral portions of the outermost annular lamella. The intradiscal pressure of the adjacent disc was significantly increased by 0.29 (0.13, 0.47) MPa after fusion (p < 0.05). This study demonstrated that fusion could cause alterations in adjacent disc biomechanics, and the combined in vivo and in silico method could be a valuable tool for the quantitative assessment of ASD after fusion.