Introduction: Cobalt ions (Co²⁺) stabilize HIFα and increase endogenous erythropoietin (EPO) production creating the possibility that Co²⁺ supplements (CoSupp) may be used as performance enhancing substances. The aim of this study was to determine the effects of a small oral dosage of CoSupp on hemoglobin mass (Hbmass) and performance with the objective of providing the basis for establishing upper threshold limits of urine [Co²⁺] to detect CoSupp misuse in sport.Methods: Twenty-four male subjects participated in a double-blind placebo-controlled study. Sixteen received an oral dose of 5 mg of ionized Co²⁺ per day for 3 weeks, and eight served as controls. Blood and urine samples were taken before the study, during the study and up to 3 weeks after CoSupp. Hbmass was determined by the CO-rebreathing method at regular time intervals, and VO_2max was determined before and after the CoSupp administration period.Results: In the Co²⁺ group, Hbmass increased by 2.0 ± 2.1% (p < 0.001) while all the other analyzed hematological parameters did not show significant interactions of time and treatment. Hemoglobin concentration ([Hb]) and hematocrit (Hct) tended to increase (p = 0.16, p = 0.1) and also [EPO] showed a similar trend (baseline: 9.5 ± 3.0, after 2 weeks: 12.4 ± 5.2 mU/ml). While mean VO_2max did not change, there was a trend for a positive relationship between changes in Hbmass and changes in VO_2max immediately after CoSupp (r = 0.40, p = 0.11). Urine [Co²⁺] increased from 0.4 ± 0.3 to 471.4 ± 384.1 ng/ml (p < 0.01) and remained significantly elevated until 2 weeks after cessation.Conclusion: An oral Co²⁺ dosage of 5 mg/day for 3 weeks effectively increases Hbmass with a tendency to increase hemoglobin concentration ([Hb]) and hematocrit (Hct). Because urine Co²⁺ concentration remains increased for 2 weeks after cessation, upper limit threshold values for monitoring CoSupp can be established.