The melanocortin neuronal system, which consists of hypothalamic proopiomelanocortin (POMC) and agouti-related protein (AgRP) neurons, is a leptin target that regulates energy balance and metabolism, but studies in humans are limited by a lack of reliable biomarkers to assess brain melanocortin activity. The objective of this study was to measure the POMC prohormone and its processed peptide, β-endorphin (β-EP), in cerebrospinal fluid (CSF) and AgRP in CSF and plasma after calorie restriction to validate their utility as biomarkers of brain melanocortin activity. CSF and plasma were obtained from 10 lean and obese subjects after fasting (40 h) and refeeding (24 h), and from 8 obese subjects before and after 6 wk of dieting (800 kcal/day) to assess changes in neuropeptide and hormone levels. After fasting, plasma leptin decreased to 35%, and AgRP increased to 153% of baseline. During refeeding, AgRP declined as leptin increased; CSF β-EP increased, but POMC did not change. Relative changes in plasma and CSF leptin were blunted in obese subjects. After dieting, plasma and CSF leptin decreased to 46% and 70% of baseline, CSF POMC and β-EP decreased, and plasma AgRP increased. At baseline, AgRP correlated negatively with insulin and homeostasis model assessment (HOMA-IR), and positively with the Matsuda index. Thus, following chronic calorie restriction, POMC and β-EP declined in CSF, whereas acutely, only β-EP changed. Plasma AgRP, however, increased after both acute and chronic calorie restriction. These results support the use of CSF POMC and plasma AgRP as biomarkers of hypothalamic melanocortin activity and provide evidence linking AgRP to insulin sensitivity.