Although a 40 % absorption of a standard reference dose corresponds to iron (Fe) absorption in borderline Fe-deficient subjects, this percentage is currently applied to all subjects independent of Fe status: (a) to assess the use of the 40 % of Fe absorption of the reference dose (FeRD%) for subjects with iron-depleted stores (IDS), normal Fe status (NIS), Fe deficiency without anemia (IDWA), and Fe deficiency anemia (IDA) and (b) to explore relationships between Fe status biomarkers and FeRD%. Six hundred forty-six participants (582 women and 64 men) were selected from multiple Fe bioavailability studies and classified into four groups based on Fe status: NIS, IDS, IDWA, and IDA. All men were classified as normal. The absorption from FeRD% was calculated in each group and correlated with Fe status biomarkers. (a) Women with IDS absorbed 40 (18.9-84.7) % of the reference dose; (b) for male subjects with NIS, the absorption of the reference dose was 19 (9.8-36.1) %, while for females, absorption was observed as to be 34 (16.7-68.6)%. In the case of subjects with IDWA, a 43 (19.7-92.5) % absorption was observed, while subjects with IDA demonstrated 67 (45.2-98.6) % absorption. Serum ferritin (SF) had the strongest inverse correlation with FeRD% (r = -0.41, p < 0.001). A transferrin saturation (TS) <15 % increases the probability that the FeRD% will be highly elevated (OR, 5.05; 95 % CI, 2.73, 9.31; p < 0.001). A 40 % absorption as reference dose is only appropriate to assess Fe absorption in subjects with IDS and IDWA. SF had an inverse correlation with FeRD%, and TS increases the probability that the FeRD% will be elevated by over fivefold.