The relationship between fasting interval and glucose screening was assessed in a prospective study of 153 non-diabetic pregnant patients undergoing a standard 50g glucose challenge test. An interval of less than 3 hours after the last meal was found to be associated with a significantly greater insulin response as opposed to a fasting interval of more than 3 hours (121.3 pmol/l vs. 83.5 pmol/l, p less than .001) and a greater insulin/glucose index (0.92 vs. 0.66, p less than .001), with no difference in plasma glucose. Overall, obese patients had a higher glucose response than non-obese, but only higher insulin levels (107 pmol/l vs. 69 pmol/l, p less than .001) and insulin/glucose index (0.79 vs. 0.59, p less than .02) when the fasting interval was greater than 3 hours. Our data suggest that the fasting interval can influence insulin response during a glucose challenge test and the aberration in insulin secretion may effect screening results, especially in the high-risk gravida with glucose abnormality.