Pre-clinical in vitro tests are needed to evaluate the biomechanical performance of new spinal implants. For such experiments large animal models are frequently used. Whether these models allow any conclusions concerning the implant’s performance in humans is difficult to answer. The aim of the present study was to investigate whether calf, pig or sheep spine specimens may be used to replace human specimens in in vitro flexibility and cyclic loading tests with two different implant types. First, a dynamic and a rigid fixator were tested using six human, six calf, six pig and six sheep thoracolumbar spine specimens. Standard flexibility tests were carried out in a spine tester in flexion/extension, lateral bending and axial rotation in the intact state, after nucleotomy and after implantation. Then, the Coflex interspinous implant was tested for flexibility and intradiscal pressure using another six human and six calf lumbar spine segments. Loading was carried out as described above in the intact condition, after creation of a defect and after implantation. The fixators were most easily implantable into the calf. Qualitatively, they had similar effects on ROM in all species, however, the degree of stability achieved differed. Especially in axial rotation, the ROM of sheep, pig and calf was partially less than half the human ROM. Similarly, implantation of the Coflex interspinous implant caused the ROM to either increase in both species or to decrease in both of them, however, quantitatively, differences were observed. This was also the case for the intradiscal pressure. In conclusion, animal species, especially the calf, may be used to get a first idea of how a new pedicle screw system or an interspinous implant behaves in in vitro flexibility tests. However, the effects on ROM and intradiscal pressure have to be expected to differ in magnitude between animal and human. Therefore, the last step in pre-clinical implant testing should always be an experiment with human specimens.