ABSTRACT Introduction Mountain ultramarathon (MUM) induces extreme physiological stress for the human body. For instance, a decrease in total hemoglobin mass (Hbmass) due to severe hemolysis is historically suspected. Nevertheless, hematological changes following a 330-km MUM have to date never been investigated. Methods Blood volumes were determined before (pre-) and after (post-) a 330-km race completed by 13 participants, through the automated carbon monoxide (CO)-rebreathing method. Native viscosity and normalized blood viscosity were determined using a cone/plate viscometer at five different speeds (11.25 to 225 s−1). Biomarkers of inflammation, erythropoiesis, and hemolysis were additionally quantified. Results Following the race, an 18% rise in plasma volume (3338 ± 568 vs 3928 ± 590 mL, P = 0.001) was observed, whereas absolute Hbmass (802 ± 102 vs 833 ± 111 g, P = 0.09) did not change significantly. A decrease in native viscosity was reported at all speeds (P < 0.001) with a significant reduction for normalized viscosity at low to intermediate speeds only (i.e., 11.25, 22.5, and 45 s−1). Marked inflammation was suggested by upregulated interleukin-6 (7.1 ± 8 vs 16.5 ± 14 ng⋅L−1, P = 0.011) and C-reactive protein levels (12.3 ± 14 vs 51.6 ± 14 μg⋅mL−1, P = 0.001). Besides, the increased erythropoietin (5.7 ± 3 vs 12 ± 6 mU⋅mL−1, P = 0.021) and erythroferrone levels (6.5 ± 4 vs 8.5 ± 4 ng⋅L−1, P = 0.001) may indicate enhanced erythropoiesis. Conclusions Overall, these findings suggest an enhanced red blood cell turnover, probably triggered by limited exercise-induced hemolysis (although still supported by the decrease in corrected viscosity), likely balanced through accelerated erythropoiesis.