How to optimise glucose metabolism in the traumatised human brain remains unclear, including whether injured brain can metabolise additional glucose when supplied. We studied the effect of microdialysis-delivered 1,2-13C2 glucose at 4 and 8 mmol/L on brain extracellular chemistry using bedside ISCUSflex, and the fate of the 13C label in the 8 mmol/L group using high-resolution NMR of recovered microdialysates, in 20 patients. Compared with unsupplemented perfusion, 4 mmol/L glucose increased extracellular concentrations of pyruvate (17%, p = 0.04) and lactate (19%, p = 0.01), with a small increase in lactate/pyruvate ratio (5%, p = 0.007). Perfusion with 8 mmol/L glucose did not significantly influence extracellular chemistry measured with ISCUSflex, compared to unsupplemented perfusion. These extracellular chemistry changes appeared influenced by the underlying metabolic states of patients' traumatised brains, and the presence of relative neuroglycopaenia. Despite abundant 13C glucose supplementation, NMR revealed only 16.7% 13C enrichment of recovered extracellular lactate; the majority being glycolytic in origin. Furthermore, no 13C enrichment of TCA cycle-derived extracellular glutamine was detected. These findings indicate that a large proportion of extracellular lactate does not originate from local glucose metabolism, and taken together with our earlier studies, suggest that extracellular lactate is an important transitional step in the brain's production of glutamine.