Objective To observe the effect of paraformaldehyde fixation on viscoelastic properties of the vertebrae in rats, so as to find the best methods of preserving cancellous bone samples from the perspective of biomechanics. Methods Twenty 8-week-old healthy female Sprague-Dawley rats were selected, and their whole L4 and L5 vertebra were separated by surgery. The total 40 vertebrae were randomly and evenly divided into experimental group and control group. The experimental group was fixed with 4% paraformaldehyde for 72 h, and the control group was transferred to 5 mL EP tube and cryopreserved at -20 ℃. Ten vertebrae were randomly selected from each group for stress relaxation and creep experiments. After 7 200 s, the samples were collected and their micro-structure changes were analyzed by micro-CT. Results The relaxation creep curve of experimental group was smoother than that of control group, the time to reach steady state was shorter, and the total amount of relaxation creep at 500 s and 7 200 s was significantly decreased (P<0.01). Micro-CT results showed that relaxation and creep experiment could cause trabecular rupture, and trabecular damage was more severe in experimental group than that in control group. Conclusions Paraformaldehyde significantly reduce the viscoelasticity of rat vertebrae, and it is more easily to cause microstructure damage under mechanical stimulation, which is detrimental to cancellous bone preservation.