The overall rate of enzymatic protein hydrolysis decreases with increasing protein concentration (0.1-30% (w/v)) at constant enzyme/substrate ratio. To understand the role of water, the amount of available water was expressed as the ratio between free and bound water and experimentally determined from water activity and T-2 relaxation time (NMR) measurements. At low protein concentrations a large excess of water is present (1.5 x 10(6) water molecules per protein molecule at 0.1% (w/v) whey protein isolate (WPI), but only 3984 at 30% (w/v) WPI. Assuming that 357 molecules of water are needed for full hydration of the protein, these values correspond to a 4280 and 11 times excess of water, showing that at 30% (w/v) WPI the amount of water becomes limited. At the same time, only a small decrease was observed in water activity (1.00-0.997 for 0.1-30% (w/v) WPI), and an increase of bound water measured by NMR (<1-10% of bound water for 0.1-30% (w/v)WPI). Additionally, the free to bound water ratio was calculated, which showed a decrease with increasing protein concentration and with addition of co-solutes (e.g. glucose, or 0.5 M NaCl). This decrease correlates with a lower initial hydrolysis rate and a lower degree of hydrolysis (DH) reached. (C) 2014 Elsevier Ltd. All rights reserved.