Abstract Advancing methodologies in high field multinuclear 1H/ 31P NMR spectroscopy continue to improve the chemical shift precision and signal amplitude sensitivity attainable in ex vivo NMR studies of respiring tissues. Thus it was straightforward in a 14.1-Tesla (600-MHz) system to use the well-known temperature dependence of the chemical shift of water protons to implement a protocol addition in our brain slice studies (350 μm thick, 15 slices per 8-mm NMR tube) of hypoxia. The protocol addition provides for accurate, continuous measurements of brain tissue temperature, a very important variable in studies of oxygen deprivation and metabolism. The basic protocol for loading and maintaining superfused respiring neonatal cerebrocortical slices, similar to protocols described previously by us and others, permitted rapid detection of glycolytic and TCA cycle activity. All brain slices have an ‘injury layer’ ≈50-μm thick from mechanical shear. In our protocol the injury layer is confined to one side of the slice, and appears to contribute minimally to NMR spectra.