Although in theory, the differences in design between fixed-core and mobile-core prostheses should influence motion restoration, in vivo kinematic differences linked with prosthesis design remained unclear. The aim of this study was to investigate the rationale that the mobile-core design seems more likely to restore physiological motion since the translation of the core could help to mimic the kinematic effects of the natural nucleus. In vivo intervertebral motion characteristics of levels implanted with the mobile-core prosthesis were compared with untreated levels of the same population, levels treated by a fixed-core prosthesis, and normal levels (data from literature). Patients had a single-level implantation at L4L5 or L5S1 including 72 levels with a mobile-core prosthesis and 33 levels with a fixed-core prosthesis. Intervertebral mobility characteristics included the range of motion (ROM), the motion distribution between flexion and extension, the prosthesis core translation (CT), and the intervertebral translation (VT). A method adapted to the implanted segments was developed to measure the VT: metal landmarks were used instead of the bony landmarks. The reliability assessment of the VT measurement method showed no difference between three observers (p < 0.001), a high level of agreement (ICC = 0.908) and an interobserver precision of 0.2 mm. Based on this accurate method, this in vivo study demonstrated that the mobile-core prosthesis replicated physiological VT at L4L5 levels but not at L5S1 levels, and that the fixed-core prosthesis did not replicate physiological VT at any level by increasing VT. As the VT decreased when the CT increased (p < 0.001) it was proven that the core mobility minimized the VT. Furthermore, some physiologic mechanical behaviors seemed to be maintained: the VT was higher at implanted the L4L5 level than at the implanted L5S1 level, and the CT appeared lower at the L4L5 level than at the L5S1 level. ROM and motion distribution were not different between the mobile-core prosthesis and the fixed-core prosthesis implanted levels. This study validated in vivo the concept that a mobile-core helps to restore some physiological mechanical characteristics of the VT at the implanted L4L5 level, but also showed that the minimizing effect of core mobility on the VT was not sufficient at the L5S1 level.