Glucose intolerance, commonly seen in the elderly, is the underlying cause of many metabolic disorders including diabetes, and is measured using the glucose tolerance test (GTT). Murine models are widely used to study the development of diabetes, obesity and cardiovascular disease during ageing. Similar to humans, the GTT is routinely used to determine tolerance to glucose in mouse models, particularly C57BL/6J. In contrast to the human situation, however, it has been observed that old mice have a better, rather than poorer, tolerance to glucose than young mice after an intraperitoneal injection of glucose (De Leon et al., 2018; Oh et al., 2016; Reynolds et al., 2019). This has been explained by adaptations in old mice, such as an increase in the size of beta cells (De Leon et al., 2018) and a rise in number of calcium receptors (Oh et al., 2016), which lead to better glucose metabolism. However, an intraperitoneal injection may be biased by possible age-related differences in glucose uptake from the peritoneum into the circulation. In humans, it has also been suggested that there are inconsistencies in results and lack of reproducibility when performing an oral GTT (Nelson, 1988), so results must be interpreted carefully. To circumvent potential age-related differences in the rate of absorption of glucose into the circulation, the best way to assess glucose tolerance is direct injection into the circulation. In line with this, it has been observed that 56% of pregnant women suggested to have abnormal glucose tolerance after an oral GTT had normal glucose tolerance when assessed with a venous GTT (Benjamin & Casper, 1966). We hypothesized that an intraperitoneal injection of glucose overestimates glucose tolerance in old but not in young mice. To investigate this, after a 16-h overnight fast male C57BL/6J mice received an intraperitoneal injection (IP: young n=7; old n=6), or an intravenous injection via the tail vein (IV: young n=7; old n=6) of glucose (2 g glucose/kg body mass). A glucometer (Glucocard X-mini plus, Japan) was used to measure blood glucose from a drop of blood taken from an incision in the tail vein at baseline (0 min) and at 15, 30, 60, 90 and 120 min after the injection. Experiments were approved by the ethics committee of the Lithuanian Republic Alimentary and Veterinary Public Office (#G2-90 in 2018).To determine the changes in blood glucose over 120 mins, a repeated-measures ANOVA with time as within factor, and age and route of injection as between factors was used. The time * route interaction in old animals (p=0.003) was reflected by higher glucose concentrations at all time points after an intravenous than an intraperitoneal injection. The absence of a time * route interaction (p=0.090) in young animals indicated that the time course of the change in glucose concentrations after IP and IV glucose injections was similar in young animals. Our data therefore indicate that especially in old animals an intraperitoneal injection of glucose overestimates glucose tolerance and that an intravenous injection of glucose is advised to determine glucose tolerance.