Background: Glucose is the main inter-organ energy supplying metabolite in humans and other vertebrates. In clinical analyses, its measurement is probably the most performed and used for diagnostic, monitoring and control of the physiological status. However, glucose chemical structure, specially its anomeric forms (ɑ/β), may deeply interfere in their own analyses, often resulting in misleading results. Methods: These effects on glucose estimation were studied by using a common glucose oxidase/peroxidase based method, in the presence or absence of added mutarotase, which speeds up the ɑ/β conversion rate. Glucose concentrations were measured in pure standards and plasma samples from control and cafeteria diet-fed rats. Results: The addition of mutarotase resulted in higher glucose readings, independently of glucose concentration and its initial anomeric proportions in the sample. In the absence of mutarotase, cafeteriafed rats had higher glucose levels than controls, but the differences disappeared in its presence, because under experimental conditions, a proportion of the α-anomer was not isomerized and thus was not measured. Conclusions: Diet altered the proportion of anomers, suggesting that glucose usage by physiological processes affects the anomers' ratio and may have an important metabolic meaning, which deserves a detailed study in addition to the need to correct the methods in use to obtain real 'total glucose' readings.