Bed rest (BR) causes bone loss, even in otherwise healthy subjects. Several studies suggest that ambulatory subjects may benefit from high-protein intake to stimulate protein synthesis and to maintain muscle mass. However, increasing protein intake above the recommended daily intake without adequate calcium and potassium intake may increase bone resorption. We hypothesized that a regimen of high-protein intake (HiPROT), applied in an isocaloric manner during BR, with calcium and potassium intake meeting recommended values, would prevent any effect of BR on bone turnover. After a 20-day ambulatory adaptation to a controlled environment, 16 women participated in a 60-day, 6 degrees head-down-tilt (HDT) BR and were assigned randomly to 1 of 2 groups. Control (CON) subjects (n = 8) received 1 g/(kg body mass . day)(-1) dietary protein. HiPROT subjects (n = 8) received 1.45 g protein/(kg body mass . day)(-1) plus an additional 0.72 g branched-chain amino acids per day during BR. All subjects received an individually tailored diet (before HDTBR: 1888 +/- 98 kcal/day; during HDTBR: 1604 +/- 125 kcal/day; after HDTBR: 1900 +/- 262 kcal/day), with the CON group's diet being higher in fat and carbohydrate intake. High-protein intake exacerbated the BR-induced increase in bone resorption marker C-telopeptide (>30%) (p < 0.001) by the end of BR. Bone formation markers were unaffected by BR and high-protein intake. We conclude that high-protein intake in BR might increase bone loss. Further long-duration studies are mandatory to show how the positive effect of protein on muscle mass can be maintained without the risk of reducing bone mineral density.