Background Deliberately training with reduced carbohydrate availability, a paradigm coined training low, has shown to promote adaptations associated with improved aerobic capacity. In this context researchers have proposed that protein may be ingested prior to training as a means to enhance the protein balance during exercise without spoiling the effect of the low carbohydrate availability. Accordingly, this is being practiced by world class athletes. However, the effect of protein intake on muscle protein metabolism during training low has not been studied. This study aimed to examine if protein intake prior to exercise with reduced carbohydrate stores benefits muscle protein metabolism in exercising and non-exercising muscles. Methods Nine well-trained subjects completed two trials in random order both of which included a high-intensity interval ergometer bike ride (day 1), a morning (day 2) steady state ride (90 min at 65% VO(2)peak, 90ss), and a 4-h recovery period. An experimental beverage was consumed before 90ss and contained either 0.5 g whey protein hydrolysate [WPH]/ kg lean body mass or flavored water [PLA]. A stable isotope infusion (L-[ring-C-13(6)]-phenylalanine) combined with arterial-venous blood sampling, and plasma flow rate measurements were used to determine forearm protein turnover. Myofibrillar protein synthesis was determined from stable isotope incorporation into the vastus lateralis. Results Forearm protein net balance was not different from zero during 90ss exercise (nmol/100 ml/min, PLA: 0.5 +/- 2.6; WPH: 1.8, +/- 3.3) but negative during the 4 h recovery (nmol/100 ml/min, PLA: - 9.7 +/- 4.6; WPH: - 8.7 +/- 6.5); no interaction (P = 0.5) or main effect of beverage (P = 0.11) was observed. Vastus lateralis myofibrillar protein synthesis rates were increased during 90ss exercise (+ 0.02 +/- 0.02%/h) and recovery (+ 0.02 +/- 0.02%/h); no interaction (P = 0.3) or main effect of beverage (P = 0.3) was observed. Conclusion We conclude that protein ingestion prior to endurance exercise in the energy- and carbohydrate-restricted state does not increase myofibrillar protein synthesis or improve net protein balance in the exercising and non-exercising muscles, respectively, during and in the hours after exercise compared to ingestion of a non-caloric control.