OBJECTIVE - We tested the hypotheses that human brain glycogen is mobilized during hypoglycemia and its content increases above normal levels ("supercompensates") after hypoglycemia. RESEARCH DESIGN AND METHODS - We utilized in vivo 13C nuclear magnetic resonance spectroscopy in conjunction with intravenous infusions of [13C]glucose in healthy volunteers to measure brain glycogen metabolism during and after euglycemic and hypoglycemic clamps. RESULTS - After an overnight intravenous infusion of 99% enriched [1-13C]glucose to prelabel glycogen, the rate of label wash-out from [1-13C]glycogen was higher (0.12 ± 0.05 vs. 0.03 ± 0.06 μmol·g-1·h-1, means ± SD, P < 0.02, n = 5) during a 2-h hyperinsulinemic-hypoglycemic clamp (glucose concentration 57.2 ± 9.7 mg/dl) than during a hyperinsulinemic-euglycemic clamp (95.3 ± 3.3 mg/dl), indicating mobilization of glucose units from glycogen during moderate hypoglycemia. Five additional healthy volunteers received intravenous 25-50% enriched [1- 13C]glucose over 22-54 h after undergoing hyperinsulinemic-euglycemic (glucose concentration 92.4 ± 2.3 mg/dl) and hyperinsulinemic- hypoglycemic (52.9 ± 4.8 mg/dl) clamps separated by at least 1 month. Levels of newly synthesized glycogen measured from 4 to 80 h were higher after hypoglycemia than after euglycemia (P ≤ 0.01 for each subject), indicating increased brain glycogen synthesis after moderate hypoglycemia. CONCLUSIONS - These data indicate that brain glycogen supports energy metabolism when glucose supply from the blood is inadequate and that its levels rebound to levels higher than normal after a single episode of moderate hypoglycemia in humans.