BackgroundPrevious studies showed that pre- and probiotics may enhance iron absorption. Probiotics combined with prebiotics (synbiotics), including human-identical milk oligosaccharides (HiMO), are commonly added to infant and follow-up formula (FUF). Whether these additions enhance iron absorption from iron-fortified commercial milk formula is uncertain. ObjectiveWe determined the effect of adding: 1) a synbiotic (galacto-oligosaccharides (GOS)+Limosilactobacillus reuteri [L. reuteri]), or 2) the HiMO 2'-fucosyllactose (2’FL) to iron-fortified FUF on iron absorption in young Thai children. MethodsIn a randomized, controlled, single-blinded (participants) cross-over study, 82 Thai children aged 8–14 months were enrolled to consume single servings (235ml) of FUF with isotopically labelled ferrous sulfate (2.2 mg iron) with: 1) the synbiotic (400 mg/100 ml GOS and L. reuteri DSM 17938); 2) the 2’FL (100 mg/100 ml); and 3) without synbiotic and without 2’FL (control) in random order and a 3-day wash-out period between administrations. Fractional iron absorption (FIA[%]) was assessed by measuring erythrocyte incorporation of isotopic labels 14 days (n=26) and 28 days (n=76) after consumption of the last test FUF. ResultsMedian (IQR) FIA from iron-fortified FUF with the synbiotic (8.2[5.2, 12.9]%) and with 2’FL (8.4[5.5, 14.1]%) did not differ from the control FUF (8.1[4.8,14.7]%) (synbiotic vs. control, P=0.24; 2’FL vs. control, P=0.95). FIA from all FUF did not differ when measured after 14 and 28 days of erythrocyte incorporation (Time, P=0.368; FUF, P=0.435; Time x FUF, P=0.937). Fecal pH and hemoglobin were negatively associated with FIA. ConclusionIn young Thai children, the addition of a synbiotic (GOS+L. reuteri) or 2’FL to iron-fortified FUF did not impact FIA from a single serving. The trial was registered at ClinicalTrial.gov as NCT04774016.The study was registered at clinicaltrials.gov as NCT04774016.