Molecular mobility in form of alpha and beta relaxations is considered crucial for characterization of amorphous lyophilizates and reflected in the transition temperatures Tg_α and Tg_β. Based on an overview of applied methods to study beta relaxations, Dynamic Mechanical analysis was used to measure Tg_α and Tg_β in amorphous freeze-dried samples. Lysozyme and trehalose as well as their mixtures in varying ratios were investigated. Three different residual moisture levels, ranging from roughly 0.5–7 % (w/w), were prepared via equilibration of the freeze-dried samples. Known plasticising effects of water on Tg_α were confirmed, also via differential scanning calorimetry. In addition and contrary to expectations, an influence of water on the Tg_β also was observed. On the other hand, an increasing amount of trehalose lowered Tg_α but increased Tg_β showing that Tg_α and Tg_β are not paired. The findings were interpreted with regard to their underlying molecular mechanisms and a correlation with the known influences of water and trehalose on stability. The results provide encouraging hints for future stability studies of freeze-dried protein formulations, which are urgently needed, not least for reasons of sustainability.