Rapid decreases in hemoglobin mass (Hbmass) have been reported in healthy humans with spaceflight and following descent from high altitude. It has been proposed that a selective increase in the destruction of young red blood cells (RBCs) mediates these decreases but conclusive evidence demonstrating neocytolysis is lacking. Based on the proposed triggers and time course of adaptation during spaceflight, we hypothesized that 4 days of −6° head-down tilt bed rest (HDTBR) would cause a rapid decrease in Hbmass that would be associated with evidence of increased RBC destruction. PURPOSE: To examine changes in Hbmass before (PRE), 5 hours after (POST), and 5 days after (POST5) 4 days of HDTBR. METHODS: Seven healthy, recreationally active men (age: 21 ± 3 years, peak oxygen uptake: 50 ± 6 mL kg-1 min-1) completed 4 days of HDTBR. Hbmass was assessed using the optimized carbon monoxide rebreathing method. Markers of RBC production and destruction assessed included [erythropoietin] ([EPO]), [soluble transferrin receptor] ([sTfr]), reticulocyte count, [ferritin], [haptoglobin], and [bilirubin]. RESULTS: [EPO] decreased by 30 ± 33% from PRE to POST (p = 0.028). Contrary to our hypothesis, Hbmass was increased by 4.0 ± 4.3% from PRE to POST (p = 0.014) before decreasing to a level 3.6 ± 2.4% below PRE at POST5 (p = 0.027). From PRE to POST, [haptoglobin] increased 66 ± 73% (p = 0.013), [bilirubin] decreased 26 ± 34% (p = 0.054), [ferritin] increased 17 ± 17 % (p = 0.012), and reticulocyte count remained stable. From PRE to POST5, sTfr decreased 17 ± 5% (p = 0.018) but there were no significant alterations in [ferritin], [haptoglobin], [bilirubin] or reticulocyte count. CONCLUSION: Our findings suggest that 4-day HDTBR results in a transient increase in Hbmass that may be influenced by decreased RBC destruction. However, since the POST measurement occurred following re-ambulation, a potential role for other factors (i.e., spleen contraction) on the increase in Hbmass cannot be excluded. The decrease in Hbmass at POST5 appears to be mediated by decreased RBC production rather than increased RBC destruction. These findings highlight the need to re-examine the time course and mechanisms of Hbmass alterations with short-term spaceflight and simulated microgravity.