Plasticizing effects of water and glycerol are widely used in pharmaceutical applications of gelatin films. We investigated gelatin films containing water or water and glycerol equilibrated under a range of relative humidity. Films were characterized by differential scanning calorimetry running in classical and modulated modes. Glass and melting transition temperatures, relaxation and melting enthalpies and heat capacity changes were determined versus film composition and maturation time. This investigation quantifies the amount of collagen type structure through the melting enthalpy of triple helices. A unified phase diagram for glass and melting transition temperatures is presented as a universal curve for any proportion of water and glycerol in films versus a single parameter, which is the ratio of hydroxyl groups belonging to water or glycerol per amino-acid group and highlights the role played by the hydrogen bonding. The molecular mechanisms involved in the plasticizing effects are discussed. (C) 2012 Elsevier Ltd. All rights reserved.