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We investigated whether immature reticulocyte fraction (IRF) and immature reticulocytes to red blood cells ratio (IR/RBC) are sensitive biomarkers for low-dose recombinant human erythropoietin (rhEpo) treatment at sea-level (SL) and moderate altitude (AL) and whether multi (FACS) or single (Sysmex-XN) fluorescence flow cytometry is superior for IRF and IR/RBC determination. Thirty-nine participants completed two interventions, each containing a four-week baseline, a four-week SL or AL (2,230m) exposure and a four-week follow-up. During exposure, rhEpo (20 IU·kg-1) or placebo (PLA) was injected at SL (SLrhEpo n = 25, SLPLA n = 9) and AL (ALrhEpo n = 12, ALPLA n = 27) every second day for three weeks. Venous blood was collected weekly. Sysmex measurements revealed that IRF and IR/RBC was up to ~70% (P < 0.01) and ~190% (P < 0.001) higher in SLrhEpo than SLPLA during treatment and up to ~45% (P < 0.001) and ~55% (P < 0.01) lower post-treatment, respectively. Compared with ALPLA, IRF and IR/RBC was up to ~20% (P < 0.05) and ~45% (P < 0.001) lower post-treatment in SLrhEpo, respectively. In ALrhEpo, IRF and IR/RBC was up to ~40% (P < 0.05) and ~110% (P < 0.001) higher during treatment and up to ~25% (P < 0.05) and ~40% (P < 0.05) lower post-treatment, respectively, compared with ALPLA. Calculated thresholds provided ~90% sensitivity for both biomarkers at SL and 33% (IRF) and 66% (IR/RBC) at AL. Specificity was >99%. Single-fluorescence flow cytometry coefficient of variation was >twofold higher at baseline (P < 0.001), and provided larger or similar changes compared to multi-fluorescence, albeit with smaller precision. In conclusion, IRF and IR/RBC were sensitive and specific biomarkers for low-dose rhEpo misuse at SL and AL.