This study was conducted as part of research line addressing the mechanical response of periodontal ligament (PDL) to tensile-compressive sinusoidal loading. The aim of the present project was to determine the effect of three potential sources of variability: (1) specimen geometry, (2) tissue preconditioning and (3) tissue structural degradation over time. For the three conditions, selected mechanical parameters were evaluated and compared. (1) Standard flat specimens (obtained by sequentially slicing portions of bone, PDL and dentin using a precision band saw) and new cylindrical specimens (extracted with a diamond-coated trephine drill) were obtained from bovine mandibular first molars and subjected to a sinusoidal load profile. (2) Specimens were loaded with up to 2000 cycles. (3) Specimens were immersed in saline and tested after 0, 30 and 60 min. From the data generated, the following was concluded: (1) specimen geometry and preparation technique do not influence the mechanical response of the PDL; (2) the mechanical response stabilizes after approximately 1000 cycles; and (3) no major structural degradation occurs when PDL is immersed in saline for a time lapse up to 60 min.