The effects of glycerol and moisture contents on the thermal transitions of whey protein isolate (WPI) powder-glycerol-water mixtures were studied. Mixtures with ratios of 100:0, 70:30, 60:40, and 50:50 WPI:glycerol on a dry basis (db) were preconditioned to 0.34 +/- 0.01 (25.4 +/- 0.4 degrees C) and 0.48 +/- 0.02 (25.9 +/- 2.2 degrees C) water activity. Differential scanning calorimetry (DSC) showed the existence of an endothermic peak starting at 148.3 +/- 0.7 degrees C for 100% WPI preconditioned to a water activity of 0.34 +/- 0.01. The onset temperature of this peak decreased with addition and increase of glycerol content, as well as with the increase in water activity from 0.34 +/- 0.01 to 0.48 +/- 0.02. An additional endothermic transition, important for extruding the mixtures into flexible sheets, occurred in mixtures containing 50% glycerol db, preconditioned to 0.48 +/- 0.02 water activity. The onset temperature of the peak was 146 +/- 2.0 degrees C. Whey protein-based sheets containing 45.8%, 48.8%, and 51.9% glycerol db were obtained using a Haake-Leistritz corotating twin-screw extruder. All samples were obtained at a screw speed of 250 rpm and a final barrel-temperature profile of 20, 20, 20, 80, 110, and 130 degrees C. Melt temperature at the time of sheet formation was 143 to 150 degrees C. Average thickness of the sheets was 1.31 +/- 0.02 mm. Samples with 45.8% glycerol db had significantly higher tensile strength (TS) than samples with higher glycerol contents. Also, as glycerol concentration increased, sheet elastic modulus (EM) decreased significantly (P= 0.05). Extrusion of whey protein-based sheets is an important step toward extrusion of thinner edible films for food wraps, layers, or pouches.