Aims: There are previous suggestions of increased C-peptide concentration in women with Type 1 diabetes during pregnancy. Our aim was to re-evaluate the hypothesis of a pregnancy-induced increase by measuring plasma C-peptide concentration in women with stable blood glucose control under standardized fasting and meal-stimulated conditions. Methods: Ten women with Type 1 diabetes; median age 31.1 years, median diabetes duration 19 years, median HbA1c 52 mmol/mol (6.9%) were admitted to a clinical research facility for two 24-h visits in early (12�16 weeks) and late (28�32 weeks) pregnancy. Women They ate standardized study meals - 80-g carbohydrate dinner, 60-g carbohydrate breakfast, and fasted between meals and overnight. Closed-loop insulin delivery maintained stable and comparable glycaemic conditions. Paired samples for plasma glucose and C-peptide were obtained. Results: Plasma glucose levels were comparable in early (median 6.5 mmol/l; interquartile range 5.6�8.6) and late pregnancy (median 7.0 mmol/l; interquartile range 6.1�7.8; P = 0.72). There was no change in fasting or meal-stimulated plasma C-peptide concentration from early to late pregnancy; mean difference 4.0 pmol/l (95% CI ?6.0 to 7.0; P = 0.9). Four women had detectable C-peptide; peak (range) early vs. late pregnancy 48.5 (10�115) vs. 40.0 pmol/l (80�105); P = 0.5, which was weakly associated with plasma glucose; R2 = 0.15, P < 0.0001. Conclusions: We found no gestational changes in plasma C-peptide concentration. Previously reported increases may reflect differences in glucose control and/or exogenous insulin doses. This study highlights the importance and challenges of standardizing experimental conditions for accurate plasma C-peptide measurement during Type 1 diabetes pregnancy.