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Purpose: Hemodilutional anemia is associated with acute kidney injury (AKI) and mortality in patients undergoing cardiac surgery by mechanisms that may include tissue hypoxia. Our hypothesis was to assess if changes in the potential hypoxic biomarkers, including methemoglobin and erythropoietin, correlated with a decrease in hemoglobin (Hb) concentration following hemodilution on cardiopulmonary bypass (CPB)., Methods: Arterial blood samples were taken from patients (n = 64) undergoing heart surgery and CPB at baseline, during CPB, following CPB, and in the intensive care unit (ICU). Potential hypoxic biomarkers were measured, including methemoglobin, plasma Hb, and erythropoietin. Data were analyzed by repeated measures one-way analysis of variance on ranks and linear regression., Results: Hemoglobin levels decreased following CPB and methemoglobin increased in the ICU (P < 0.001 for both). No correlation was observed between the change in Hb and methemoglobin (P = 0.23). By contrast, reduced Hb on CPB correlated with increased lactate, reduced pH, and increased erythropoietin levels following CPB (P ≤ 0.004 for all). Increased plasma Hb (P < 0.001) also correlated with plasma erythropoietin levels (P < 0.001)., Conclusion: These data support the hypothesis that erythropoietin rather than methemoglobin is a potential biomarker of anemia-induced tissue hypoxia. The observed relationships between decreased Hb during CPB and the increase in lactate, reduced pH, and increase in erythropoietin levels suggest that early changes in plasma erythropoietin may be a pragmatic early biomarker of anemia-induced renal hypoxia. Further study is required to determine if anemia-induced increases in erythropoietin may predict AKI in patients undergoing cardiac surgery., Trial Registration: www.clinicaltrials.gov (NCT01883713). Registered 21 June 2013.