The aims of this study were to compare different tracer methods to assess whole body protein turnover, during 6h of prolonged endurance exercise when carbohydrate was ingested throughout and to investigate whether the addition of protein can improve protein balance. Eight endurance trained athletes were studied on 2 different occasions at rest (4h), during 6h of exercise at 50% VO₂max (in sequential order: 2.5h of cycling, 1h running and 2.5h of cycling) and during subsequent recovery (4h). Subjects ingested either carbohydrate (CHO trial; 0.7 g CHO^.kg^-1.h^-1) or carbohydrate/protein beverages (CHO+PRO trial; 0.7 g CHO^.kg^-1.h^-1 and 0.25 g PRO^.kg^-1.h^-1) at 30 min intervals during the entire study. Whole body protein metabolism was determined by infusion of L-[1-¹³C]leucine, L-[²H₅]phenylalanine and [¹⁵N₂]urea tracers with sampling of blood and expired breath. Leucine oxidation increased from rest to exercise (CHO: 27±2.5 vs. 74±8.8; CHO+PRO: 85±9.5 vs. 200±16.3 mg protein^.kg^-1.h^-1; P<0.05), whereas phenylalanine oxidation and urea production did not increase with exercise. Whole body protein balance during exercise with carbohydrate ingestion was negative (-74±8.8; -17±1.1; -72±5.7 mg protein^.kg^-1.h^-1, when using L-[1- ¹³C]leucine, L-[²H₅]phenylalanine and [¹⁵N₂]urea as tracer). Addition of protein to the carbohydrate drinks resulted in a positive or less negative protein balance (-32±16.3; 165±4.6; 151±13.4 mg protein^.kg^-1.h^-1, when using L-[1-¹³C]leucine, L-[²H₅]phenylalanine and [¹⁵N₂]urea as tracer). We conclude that even during 6h of exhaustive exercise in trained athletes using carbohydrate supplements, net protein oxidation does not increase, compared with the resting state and/or post-exercise recovery. Furthermore, the combined ingestion of both protein and carbohydrate improves net protein balance at rest as well as during exercise and post-exercise recovery.