The metabolism of glucose is one of the most fundamental ones in the cell. Indeed, glucose is the primary substrate of glycolysis and of the pentose phosphate pathway (PPP), both known to involve complex and regulated networks of enzymatic reactions. Importantly, past studies tend to suggest that the two anomers of glucose are involved in different ways, glycolysis specifically targetting the α anomer of glucose, whereas only its β anomer enters the PPP. Both glucose and glucose-6-phosphate (G6P) undergo spontaneous mutarotation α ⇌ β, a process that has been the subject of many studies. 11;12;13 However, anomeric exchange for G6P takes place on a time scale that has been shown to be two orders of magnitude faster than for glucose. The latter has a typical exchange time on the order of tens of minutes, whereas for G6P, α/β equilibrium is reached on a time scale of the order of the minute. This has direct consequences for the kinetic study of fast enzymatic reactions performed in conditions where the reaction and the mutarotation time scales overlap. Thus, in the case of glucose, spontaneous anomer exchange can be disregarded when studying the kinetics of reactions that are completed within a few tens of seconds, the typical time window of dDNP. In contrast, G6P mutarotation is much faster, which makes the catalytic dehydrogenation by the glucose-6-phosphate dehydrogenase (G6PDH) more complex to analyze, as both processes occur on overlapping time scales, and therefore cannot be observed independently. This is also the case for other enzymes targeting selectively one of the G6P anomers, such as the α-anomer specific G6P isomerase in yeast. Thus, dDNP investigations of fast enzyme reactions involving G6P as the substrate require to take both catalysis and anomer exchange into account, and to analyze them simultaneously. Such a combination of fast processes introduces further complexity, as we recently noticed in a previous dDNP study of enzymatic reactions and cascades thereof. Besides, a recent dDNP study of the G6P anomer exchange kinetics was conducted using the enzymatic phosporylation of glucose by Hexokinase. In this article, we present a kinetic study of the G6P oxidation by the enzyme G6P dehydrogenase (G6PDH), the first enzyme of the PPP that produces δ-phospho-gluconolactone, using dDNP. We use this deceptively simple example to discuss some important methodological aspects of dDNP kinetic experiments, such as its robustness and its ability to provide reproducible results, as well as the capacity of this time resolved methodology to test kinetic models and hypotheses, and to provide reliable parameter estimates. Such aspects have apparently been somewhat neglected so far.