This study validated bioelectrical impedance spectroscopy (BIS) with Cole-Cole modeled measurements of calf and arm segmental water volume and volume changes during 72 hours of simulated microgravity and caloric restriction using magnetic resonance imaging (MRI) muscle volume as a criterion method. MRI and BIS measurements of calf and upper arm segments were made in 18 healthy males and females (mean±SD, age: 29±8 y, height: 171±11 cm, mass: 71±16 kg) before and after the intervention. Muscle volume of arm and leg segments by MRI was on average 15±10% and 14±8% lower (respectively) than the estimated total water volume by BIS (P<0.01), but their correlations were excellent (r=0.96 and r=0.93, respectively). MRI vs. BIS predicted volume changes were 49±68 cc vs. 41±62 cc decrease in the calf and 18±23 cc vs. 11±24 cc decrease in the arm, respectively (P>0.05 for both). BIS detected the extracellular water shifts in the calf resulting from the head down tilt treatment, but the underfeeding protocol was not of sufficient duration or intensity to produce limb intracellular water changes detectable by BIS. BIS was highly correlated with segmental muscle volume and tracked changes associated with head down tilt. Further research, however, is needed to determine if BIS can accurately access separate changes in intracellular and extracellular volume.