Inconsistent data regarding the ability of primary tissue to detect physiological falls in glucose concentration may be due, in part, to the time-dependent ability of a cell in culture to gain energy via oxidative phosphorylation, making it more sensitive to limitations in glucose supply. Thus, hypoglycaemia may induce a non-specific stress response in these cells that could be misinterpreted as low glucose sensing. Human umbilical vascular endothelial cells (HUVEC) were enzymically and mechanically isolated from umbilical cords within 12 hours of delivery and distributed at high density onto 1% gelatin coated cover slips (Marin et al, 2001). Cells were cultured in Lonza Medium 199 containing foetal calf serum supplemented with penicillin, streptomycin and fungizone at 37°C in a 5% CO2 incubator, changing medium at every 3 days. Intracellular calcium concentration was measured by ratiometric (340/380nm) labelling with Fura 2 and the response to reducing the superfusate glucose concentration from 10mM to 0mM determined at 1, 2, 4 and 8 days after culture and compared to the response to 10uM ATP at the same time periods. Five cultures, each from 2 umbilical cords, were established and measurements made on 4 cover slips on each day of study. Time dependent differences in calcium response were analyzed by single factor ANOVA. Sheffe post hoc tests were performed as appropriate and significance was taken as P<0.05. Local ethical approval and informed consent were obtained. A significant time dependent effect was observed in response to both falls in glucose and addition of ATP (P<0.001 and P<0.002 respectively) but the pattern of response differed (P<0.0001, two way ANOVA). The calcium response to zero glucose or ATP at day 1 was low (ratiometric mean±SEM: 0.005±0.003 and 0.004±0.004 respectively). At day 2 the response to zero glucose was significantly increased by a factor of 7.4 (0.037±0.008) but although the mean response to ATP was also increased by around the same amount, to 0.033±0.020, this did not reach significance. At day 4 the response to zero glucose was sustained (0.022±0.008) while the response to ATP (0.238±0.083) was substantially and significantly increased by almost 60-fold compared to day one. On the eighth day of culture, the response to glucose dropped to a level statistically similar to that of day 1 (0.004±0.002) while the ATP response was sustained at a level not significantly different to that on day 4 (0.161±0.035). Our data shows that cultured HUVEC show a time-dependent response to the stress of hypoglycaemia following isolation and culture that differs in time course and magnitude from the response to ATP. What the mechanism involved is and whether it underlies the variability of the response to hypoglycaemia reported previously in the literature (Kumar, 2007) is not known.