Lactate is shuttled between organs, as demonstrated in the Cori cycle. Although the brain releases lactate at rest, during physical exercise there is a cerebral uptake of lactate. Here, we evaluated the cerebral lactate uptake and release in hypoxia, during exercise and when the two interventions were combined. We measured cerebral lactate turnover via a tracer dilution method ([1-13C]lactate), using arterial to right internal jugular venous differences in 9 healthy individuals (5 males and 4 females), at rest and during 30 min of submaximal exercise in normoxia and hypoxia (FiO2 10%, arterial oxygen saturation 72±10%, mean±SD). Whole-body lactate turnover increased 3.5-fold and 9-fold at two workloads in normoxia and 18-fold during exercise in hypoxia. Although middle cerebral artery mean flow velocity increased during exercise in hypoxia, calculated cerebral mitochondrial oxygen tension decreased by 13 mmHg (P<0.001). At the same time, cerebral lactate release increased from 0.15 ± 0.1 to 0.8 ± 0.6 mmol min−1 (P<0.05), corresponding to ∼10% of cerebral energy consumption. Concurrently, cerebral lactate uptake was 1.0 ± 0.9 mmol min−1 (P<0.05), of which 57 ± 9% was oxidized, demonstrating that lactate oxidation may account for up to ∼33% of the energy substrate used by the brain. These results support the existence of a cell-cell lactate shuttle that may involve neurons and astrocytes.—Overgaard, M., Rasmussen, P., Bohm, A. M., Seifert, T., Brassard, P., Zaar, M., Homann, P., Evans, K. A., Nielsen, H. B., Secher, N. H. Hypoxia and exercise provoke both lactate release and lactate oxidation by the human brain.