Background: Blood donation is associated with a loss of hemoglobin (Hb)-bound iron. Hb levels recover relatively fast by using stored iron. However, it takes more time to replenish iron stores, potentially resulting in iron deficiency.Study design: Hb and ferritin levels were measured in 5056 new, first-time, and repeat whole blood donors. We investigated whether increasing numbers of donations are associated with lower ferritin levels. Furthermore, we tested whether low ferritin levels are associated with low-Hb deferral at the subsequent donation attempt by performing logistic regression adjusted for age and stratified by sex. Results: Whereas mean Hb levels are relatively stable, ferritin levels significantly decrease with increasing numbers of donations and were approximately 50% lower for donors with >50 donations compared with those with 2-10 donations. Despite the poor correlation of ferritin and Hb levels, cross-sectional, iron-deficient donors (ferritin <15 ng/ml) had 21.8 (8.5-55.6) higher odds in men, 10.1 (6.1-16.5) in premenopausal women, and 11.7 (5.2-26.4) in postmenopausal women for Hb deferral at a subsequent visit.Discussion: To conclude, repeated donations may induce iron deficiency, which corresponds with an over tenfold increased risk of having insufficiently restored Hb levels at a subsequent donation attempt. Longer donation intervals and/or higher dietary or supplemental iron intake are warranted to prevent accumulated iron depletion and subsequent low-Hb deferral in whole blood donors.