Iron deficiency in pregnancy remains a global health burden, yet pregnancy-related physiological adaptations complicate the interpretation of iron biomarkers. Modified oral iron regimens are increasingly used in pregnancy, balancing iron intake, fractional absorption, and side effects. Non-daily dosing may improve tolerability but provides less iron. This study aimed to evaluate how daily, alternate-day, and three-times-weekly oral iron supplementation affects iron status biomarkers in non-anaemic pregnant participants. The PANDA dose-finding trial randomised non-anaemic pregnant women to 200 mg ferrous sulphate daily, on alternate days, or three-times-weekly. Blood samples were collected at 9-13 and 26-30 weeks' gestation (N=135 participants). Hepcidin and soluble transferrin receptor (sTfR) were measured by ELISA; serum iron, ferritin, transferrin, and CRP by automated biochemistry. Linear mixed-effects models assessed within- and between-group biomarker changes. Hepcidin, ferritin, and transferrin saturation declined significantly between baseline and follow-up in all groups (p<0.001). sTfR increased in the alternate-day (p=0.003) and three-times-weekly groups (p<0.001), but not in the daily group (p=0.084). Despite receiving prophylactic supplementation and maintaining hemoglobin levels, 69-72% of women were iron-deficient (ferritin <30 µg/L) at 26-30 weeks' gestation. Iron status declined across pregnancy regardless of supplementation regimen, likely reflecting dominance of physiological adaptations over iron-dosing effects. The absence of a significant rise in sTfR in the daily group suggests that daily dosing best meets the increasing iron demand of pregnancy, though confirmation in larger, adequately powered studies is required. The results further highlight the need for pregnancy-specific, functionally validated reference ranges for iron biomarkers. ISRCTN12911644.