PurposeAssessing the effects of excessive weight gain before pregnancy, in the first and second trimesters and in the month preceding glucose challenge test (GCT) on GCT results and gestational diabetes mellitus (GDM).Study designThis prospective cohort study evaluated 1279 pregnant women who were referred for their first prenatal visit in 2012–2015. Mother's body mass index (BMI) was recorded before pregnancy, during the first visit and every 4 weeks until 28 gestational weeks. All mothers underwent GCT at 28 weeks and when 1 h glucose ≥140 mg/dL (≥7.8 mmol/L), they were referred for a 100 g fasting glucose 3 h glucose tolerance test.ResultsObesity and being overweight prior to pregnancy were associated with 2.8-fold and 1.5-fold higher rates of developing GDM (p<0.001, p=0.04) and 1.9-fold and 1.8-fold higher rates of having false-positive GCT results (p<0.001). First-trimester excessive weight gain was significantly associated with false-positive GCT in women who were lean, overweight and obese before pregnancy (all p<0.001). When these women kept gaining excessive weight during the subsequent period the risk of developing GDM was significantly increased regardless of their pre-pregnancy BMI (p=0.03). When these women adhered to the recommended weight gain during the subsequent period, the risk of developing GDM was not increased, however the risk of having false-positive GCT remained high (p<0.001).ConclusionsElevated pre-pregnancy BMI independently increases the risk of GDM and false-positive GCT. First trimester weight gain is the most important predictor of GCT and GDM regardless of pre-pregnancy BMI. The weight gain during the subsequent period affects the risk of developing GDM only in women with excessive first-trimester weight gain.