Abstract : The apprehension of the factors that affect long term regulation of energy balance is indispensable to understand the rise in obesity prevalence as well as to delineate levers to prevent it. Accurate measurements of energy balance are however challenging during free-living conditions. Recent studies proposed urinary C-peptide, a metabolic byproduct of insulin synthesis, as reliable noninvasive assessment of energy balance. These studies were in fact essentially based on correlations between urinary C-peptide and energy intake and only focused on nonhuman primates. During a bed-rest study conducted in 16 healthy women in a controlled environment, we tested the existence of a relationship between 24 h-urinary C-peptide and energy balance in humans. Daily energy intake and body mass, body composition (dual-energy X-ray absorptiometry (DXA)) and total energy expenditure (doubly labeled water (DLW) method) was measured and energy balance was calculated as the difference between energy intake and expenditure. Urinary C-peptide was positively correlated with bed-rest-induced changes in fat mass (r(2) = 0.285; P = 0.03) and energy balance assessed at the end of the bed-rest (r(2) = 0.302; P = 0.027). However, in this tightly controlled environment, urinary C-peptide only accounted for 30% of variations in energy balance. No relationship was noted between urinary C-peptide and body or fat mass both at baseline and at the end of the bed-rest. These results indicate that urinary C-peptide cannot be used as an accurate biomarker of energy balance in the general human population in free-living conditions.