It is shown that the onset temperature and the magnitude of thermal events observed during DSC analyses of α-lactose monohydrate can be strongly affected by various treatments such as ageing, manual grinding and preheating (cycle of preliminary dehydration and rehydration). In the case of grinding and preheating, the change of dehydration pathways was further investigated by using a suitable combination of characterization techniques, including X-ray powder diffraction (XRPD) performed with a synchrotron source (allowing an accurate Rietveld analysis), scanning electron microscopy (SEM), laser particle size measurements, FTIR spectroscopy and 1H NMR for the determination of β-lactose contents in samples. It appeared that the dehydration mechanism is affected not only by a smaller particle size distribution, but also by residual anisotropic lattice distortions and by the formation of surface defects or high energy surfaces. The fusion–recrystallization process occurring between anhydrous forms of α-lactose at ca. 170 °C is not significantly affected by grinding, whereas a preheating treatment induces an unexpected large increase of the enthalpy associated with this transition. Our observations and interpretations confirm the important role of water molecules in the crystal cohesion of the title compound and illustrate the necessity to consider the history of each sample for a satisfactory understanding of the physical properties and the behaviour of this important pharmaceutical excipient.