Purpose Disaccharides such as trehalose are widely used as cryo-protectants to maintain the activity of proteinaceous drugs during freezing. One unresolved issue is the double transition that is observed very commonly in DSC experiments on disaccharide solutions in the frozen state; the assignment of these transitions remains disputed. Here we use calorimetry and two new techniques to shed light on the true nature of these transitions. Methods Modulated Temperature DSC (MTDSC), cryo atomic force microscopy (AFM) and a novel DMA technique were used to study these transitions. Results MTDSC identified the two transitions Tr1 and Tr2 at -35.4 and -27.9°C respectively in the reversing heat flow signal, an exotherm and endotherm were observed in the non-reversing signal at circa -32 and -29°C respectively. It is shown for the first time that AFM images can be obtained of a softening and melting sample without damaging it. A force modulation imaging technique showed a softening at Tr1 and a loss of ice crystals at Tr2. These observations were supported by the DMA results. Conclusions The results indicate Tr1 is associated with a glass transition while Tr2 is associated with the onset of loss of crystallinity.