Current markers of iron deficiency (ID) such as ferritin and hemoglobin have shortcomings, and hepcidin and erythroferrone (ERFE) could be of clinical relevance in relation to early assessment of ID. Here, we evaluate whether exposure to altitude-induced hypoxia (2,320 m) alone, or in combination with recombinant human erythropoietin (rHuEPO) treatment, affects hepcidin and ERFE levels before alterations in routine ID biomarkers and stress erythropoiesis manifest. Two interventions were completed, each comprising a four-week baseline, a four-week intervention at either sea level or altitude, and a four-week follow-up. Participants (n=39) were randomly assigned to 20 IU·kg bw rHuEPO or placebo injections every second day for three weeks during the two intervention periods. Venous blood was collected weekly. Altitude increased ERFE (P≤0.001) with no changes in hepcidin or routine iron biomarkers, making ERFE of clinical relevance as an early marker of moderate hypoxia. rHuEPO treatment at sea level induced a similar pattern of changes in ERFE (P<0.05) and hepcidin levels (P<0.05), demonstrating the impact of accelerated erythropoiesis and not of other hypoxia-induced mechanisms. Compared to altitude alone, concurrent rHuEPO treatment and altitude exposure induced additive changes in hepcidin (P<0.05) and ERFE (P≤0.001) parallel with increases in hematocrit (P<0.001), demonstrating a relevant range of both hepcidin and ERFE. A poor but significant correlation between hepcidin and ERFE was found (R=0.13, P<0.001). The findings demonstrate that hepcidin and ERFE are more rapid biomarkers of changes in iron demands than routine iron markers. Finally, ERFE and hepcidin may be sensitive markers in an anti-doping context.