Signaling pathways sense local and systemic signals and regulate muscle hypertrophy. The effects of whey protein ingestion on acute and long-term signaling responses of resistance exercise are not well known. Previously untrained young men were randomized into protein (n=9), placebo (n=9) and control (n=11) groups. Vastus lateralis (VL) muscle biopsies were taken before, and 1h and 48h after a leg press of 5x10 repetitions (RE) and after 21-wk (2x/wk) resistance training (RT). Protein (15g of whey) or non-energetic placebo was ingested before and after a single RE bout and each RE workout throughout the RT. Protein group increased its body mass and VL muscle thickness (measured by ultrasonography) already at week 10.5 (P<0.05). At week 21, protein and placebo groups had similarly increased their myofiber size. No changes were observed in the non-exercised controls. However, the phosphorylation of p70S6K and rpS6 were increased at 1h post-RE measured by western blotting, the former being the greatest with protein ingestion. mTOR phosphorylation was increased after the RE bout and RT only in the protein group whereas the protein ingestion prevented the post-RE decrease in p-4E-BP1. Akt phosphorylation decreased after RT whereas no change was observed in p-eEF2. A post-RE decrease in muscle myostatin protein occurred only in the placebo group. The results indicate that resistance exercise rapidly increases mTOR signaling and may decrease myostatin protein expression in muscle, and that whey protein increases and prolongs the mTOR signaling response.