The microstructure and properties of dentine are principal determinants of nearly all procedures in restorative dentistry. Sample sterilisation and storage are critical as the dentine sample might be affected by chemical reactions such as mineral dissolution, denaturation, and dehydration, which can lead to modification of its properties and potentially invalidate research results. This research investigates the effect of pre-test sterilisation and storage on the chemical and mechanical properties of human dentine and its response to demineralisation and fluoride treatment. According to the current study design, the mechanical properties were analysed by assessment of push-out dentine bond strength, root susceptibility to vertical fracture, dentine microhardness, nanohardness and elastic modulus, while the chemical properties were examined using scanning electron microscopy equipped with energy dispersive X-ray spectroscopy at low vacuum, inductively coupled plasma atomic emission spectroscopy and ion chromatography. Autoclave sterilisation, formalin sterilisation/storage, freezing, water and chloramine-T tooth storage at 4Â°C without changing the storage solutions did not significantly affect the push-out bond strength or root susceptibility to fracture. However, a second cycle of autoclaving significantly compromised the root resistance to fracture. In relation to sampleâs state in storage, whether whole tooth or disc, a significant reduction in dentine microhardness was caused by sterilisation/storage except for formalin and frozen disc storage, while whole tooth storage in a freezer, water or chloramine-T solutions caused significant reductions. However, autoclaving and storage of dentine as discs in water or chloramine-T caused greater reductions. The effect of sterilisation, storage and second cycle of freezing and thawing was evaluated by measuring the dentine nanohardness, elastic modulus and microhardness before and after treatment to manage the issue of variation in dental substrate properties from tooth to tooth and from one site to another on the same tooth. All the methods tested caused significant reductions in these properties except formalin, while one cycle freezing and thawing significantly reduced the nanohardness only. In relation to the chemical properties, autoclaving did not influence the dentine elemental composition or dentine response to acid etching, while tooth storage modified the dentine composition. The presence of PO43- or an alkaline storage solution did not prevent Ca2+ dissolution. Water storage produced an inorganic-deficient dentine matrix which, under acid challenge, liberated a lesser amount of PO43- than the dentine of the control group, while the frozen and chloramine-T stored dentine liberated greater amounts of minerals. Formalin-stored dentine had a higher P wt% than that of the control dentine and liberated more PO43- by acid etching. All the tested groups showed less fluoride uptake than the control group, and the formalin and freeze groups showed the closest results to that of the control.