Introduction: Fluid overload is a major challenge in hemodialysis patients and might cause hypervolemia. We speculated that hemodialysis patients reaching dry weight could have undetected hypervolemia and low hemoglobin (Hb) concentration (g/dL) due to hemodilution. Methods: The study included hemodialysis patients (n = 22) and matched healthy controls (n = 22). Blood volume, plasma volume, red blood cell volume, and total Hb mass were determined using a carbon monoxide (CO)-rebreathing method in hemodialysis patients reaching dry weight and controls. Blood volume measurements were also obtained by a dual-isotope labeling technique in a subgroup for validation purposes. Findings: In the hemodialysis group, the median specific blood volume was 89.3 mL/kg (interquartile range [IQR]: 76.7–95.4 mL/kg) and was higher than in the control group (79.9 mL/kg [IQR: 70.4–88.0 mL/kg]; p < 0.037). The median specific plasma volume was 54.7 mL/kg (IQR: 47.1–61.0 mL/kg) and 44.0 mL/kg (IQR: 38.7–49.5 mL/kg) in the hemodialysis and control groups, respectively (p < 0.001). Hb concentration was lower in hemodialysis patients (p < 0.001), whereas no difference in total Hb mass was observed between groups (p = 0.11). A correlation was found between blood volume measured by the CO-rebreathing test and the dual-isotope labeling technique in the control group (r = 0.83, p = 0.015), but not the hemodialysis group (r = 0.25, p = 0.60). Discussion: The hemodialysis group had increased specific blood volume at dry weight due to high plasma volume, suggesting a hypervolemic state. However, correlation was not established against the dual-isotope labeling technique underlining that the precision of the CO-rebreathing test should be further validated. The total Hb mass was similar between hemodialysis patients and controls, unlike Hb concentration, which emphasizes that Hb concentration is an inaccurate marker of anemia among hemodialysis patients.