Prescription of an appropriate dialysis target weight (dry weight) requires accurate evaluation of the degree of hydration. The aim of this study was to investigate whether a state of normal hydration (DW(cBIS)) as defined by calf bioimpedance spectroscopy (cBIS) and conventional whole body bioimpedance spectroscopy (wBIS) could be characterized in hemodialysis (HD) patients and normal subjects (NS). wBIS and cBIS were performed in 62 NS (33 m/29 f) and 30 HD patients (16 m/14 f) pre- and post-dialysis treatments to measure extracellular resistance and fluid volume (ECV) by the whole body and calf bioimpedance methods. Normalized calf resistivity (ρ(N)(,5)) was defined as resistivity at 5 kHz divided by the body mass index. The ratio of wECV to total body water (wECV/TBW) was calculated. Measurements were made at baseline (BL) and at DW(cBIS) following the progressive reduction of post-HD weight over successive dialysis treatments until the curve of calf extracellular resistance is flattened (stabilization) and the ρ(N)(,5) was in the range of NS. Blood pressures were measured pre- and post-HD treatment. ρ(N)(,5) in males and females differed significantly in NS. In patients, ρ(N)(,5) notably increased with progressive decrease in body weight, and systolic blood pressure significantly decreased pre- and post-HD between BL and DW(cBIS) respectively. Although wECV/TBW decreased between BL and DW(cBIS), the percentage of change in wECV/TBW was significantly less than that in ρ(N)(,5) (-5.21 ± 3.2% versus 28 ± 27%, p < 0.001). This establishes the use of ρ(N)(,5) as a new comparator allowing a clinician to incrementally monitor removal of extracellular fluid from patients over the course of dialysis treatments. The conventional whole body technique using wECV/TBW was less sensitive than the use of ρ(N)(,5) to measure differences in body hydration between BL and DW(cBIS).