Deuterium metabolic imaging (DMI) is a novel, non-invasive method to map metabolism from deuterated substrates in 3D. The replacement of protons with deuterons could potentially lead to kinetic isotope effects (KIEs), and loss of deuterons. Knowledge of the KIE levels, and label losses is required for DMI-based measurements of absolute metabolic rates. Here the deuterium KIE and label loss is investigated for glucose and acetate in rat brain in vivo using a double substrate/double labeling strategy. Significant, but predictable and reproducible label losses were observed in the metabolic products lactate, glutamate and glutamine. The measured KIE was relatively small (4-6%).