Physical hardening is a phenomenon that takes place at low temperatures, producing time-dependent isothermal changes in the rheological properties of bituminous binders. Stiffening effects caused by physical hardening combine with those consequent to chemical ageing, thus promoting the build-up of thermally-induced stresses which may lead to premature pavement failure. The main goal of this paper was to investigate the influence of physical hardening on low temperature properties of bituminous binders tested in different ageing conditions. Moreover, reversibility of hardening phenomena was directly assessed by means of a dedicated testing protocol. The study, carried out by making use of the Bending Beam Rheometer (BBR), considered four binders of different type and origin (two neat bitumens and two commercial SBS polymer-modified binders) and three ageing conditions (original, short-term ageing and long-term ageing). Obtained results, expressed in terms of a Physical Hardening Rate (PHR), showed a significant effect of chemical ageing on low temperature hardening of binders. In particular, rate of hardening was found to vary with temperature and to decrease with degree of ageing. Reversibility of the hardening process was also verified.