The aim of this study was to investigate the association between fasting duration before screening with 50g glucose challenge test (GCT) and the test outcome. For this cross-sectional study, we enrolled 508 low-risk pregnant women who underwent 50g GCT between the 24 and 28 weeks of gestation. We excluded women with pregestational diabetes, multiple gestations or a history of gestational diabetes mellitus (GDM), and macrosomia. We evaluated fasting durations, GCT results, and demographic features. A significant positive correlation was found between fasting duration and 50g GCT values (r=0.122; p=0.006), and the best cut-off value was found to be 6.5h, with 85.85% sensitivity and 38.61% specificity (relative risk, 2.73; 95% CI, 1.893-3.936; p<0.0001). Further, we divided the patients into two groups: study (fasting, <6.5h; n=146) and control (fasting,>6.5h; n=362) groups. Notably, the mean glucose levels, number of patients with GCT>140mg/dl, and rates of unnecessary 100g loadings were significantly higher in the study group. We found no significant differences between the groups in terms of the fasting plasma glucose levels and GDM prevalence. According to our findings, fasting duration of>6.5h resulted in 2.7 times more unnecessary 100g glucose tolerance tests (GTT). We recommend that patients having fasted for>6.5h receive a one-step 75g GTT after completing 8-h fasting.