Protein-based and other hydrophilic thin films are promising materials for the manufacture of edible food packaging and other food and non-food applications. Calcium caseinate (CaCas) films are highly hygroscopic and physical characterization under broad environmental conditions is critical to application development and film optimization. A new technology, humidity-controlled dynamic mechanical analysis (DMA-RH) was explored to characterize CaCas/glycerol films (3:1 ratio) during isohume temperature (T
) ramps and steps, and isothermal RH ramps and steps, to determine their mechanical and moisture-sorption properties during extensive T
and RH variations. When RH and/or T
increased, CaCas/Gly films became strongly plasticized and underwent several primary and secondary humidity-dependent transition temperatures (or transition humidities); the CaCas/Gly network hypothetically rearranged itself to adapt to the increased water-content and heat-induced molecular mobility. Between 5–40 °C and 20%–61% RH, moisture-sorption was rapid and proportional to humidity between transition points and accelerated greatly during transitions. CaCas/Gly films seemed unsuitable for storage or utilization in warm/humid conditions as they lost their mechanical integrity around Tm
~ 40 °C at 50% RH and Tm
decreased greatly with increased RH. However, below Tm
, both moisture- and heat-induced structural changes in the films were fully reversible and casein films may withstand a variety of moderate abuse conditions.