All 13C NMR studies of brain glycogen to date relied on observing the incorporation of 13C label into glycogen, and thus interpretation was potentially affected by changes in 13C label turnover rates. The goal of this study was to quantify total brain glycogen concentration under conditions of hypoglycemia or normoglycemia using biochemical methods. Rats were sacrificed using a focused microwave fixation device. The results showed that metabolism of brain glycogen was Glc- and insulin-sensitive and that insulin-induced hypoglycemia promoted a gradual glycogenolysis. Moreover, we show that there are very mild effects of isoflurane and α-chloralose anesthesia on brain glycogen concentration. Altogether these results show that total brain glycogen serves as a substantial source of glucosyl units during insulin-induced moderate hypoglycemia and therefore may be neuroprotective. Finally we also conclude that previous interpretation of 13C NMR spectroscopy data accurately reflected the changes in total brain glycogen content. © 2006 Elsevier Ltd. All rights reserved.