Background Blood donors are at risk of iron deficiency. We evaluated the effects of blood donation intensity on iron and hemoglobin in a prospective study. Methods Four cohorts of frequent and first time or reactivated blood donors (no donation in 2 years), female and male, totaling 2425 were characterized and followed as they donated blood frequently. At enrollment and the final visit, ferritin, soluble transferrin receptor (sTfR), and hemoglobin were determined. Models to predict iron deficiency and hemoglobin deferral were developed. Iron depletion was defined at two levels: Iron Deficient Erythropoiesis (IDE) [log (soluble transferrin receptor/ferritin ≥ 2.07)] and Absent Iron Stores (AIS) (ferritin < 12 ng/mL). Results Among returning female first time/reactivated donors, 20% and 51% had AIS and IDE at their final visit, respectively; corresponding proportions for males were 8% and 20%. Among female frequent donors who returned, 27% and 62% had AIS and IDE, respectively, while corresponding proportions for males were 18% and 47%. Predictors of IDE and/or AIS included a higher frequency of blood donation in the last 2 years, a shorter interdonation interval, and being female and young; conversely, taking iron supplements reduced the risk of iron depletion. Predictors of hemoglobin deferral included female gender, Black race and a shorter interdonation interval. Conclusions There is a high prevalence of iron depletion in frequent blood donors. Increasing the interdonation interval would reduce the prevalence of iron depletion and hemoglobin deferral. Alternatively, replacement with iron supplements may allow frequent donation without the adverse outcome of iron depletion.