► l-Isoleucine increased GLUT-4 translocation to the plasmatic membrane. ► l-Leucyl-l-isoleucine increased GLUT-4 translocation to the plasmatic membrane. ► l-Leucyl-l-isoleucine caused the highest insulinotropic effect. ► l-Isoleucine reduced the blood glucose levels, without increasing the insulin levels. Whey protein hydrolysate (WPH) is capable of increasing muscle glycogen reserves and of concentrating the glucose transporter in the plasma membrane (PM). The objective of this study was to determine which WPH components could modulate translocation of the glucose transporter GLUT-4 to the PM of animal skeletal muscle. Forty-nine animals were divided into 7 groups (n=7) and received by oral gavage 30% glucose plus 0.55g/kg body mass of the following WPH components: (a) control; (b) WPH; (c) l-isoleucine; (d) l-leucine; (e) l-leucine plus l-isoleucine; (f) l-isoleucyl-l-leucine dipeptide; (g) l-leucyl-l-isoleucine dipeptide. After receiving these solutions, the animals were sacrificed and the GLUT-4 analysed by western blot. Additionally, glycogen, glycaemia, insulin and free amino acids were also determined by standard methods. Of the WPH components tested, the amino acid l-isoleucine and the peptide l-leucyl-l-isoleucine showed greater efficiency in translocating GLUT-4 to the PM and of increasing glucose capture by skeletal muscle.