BACKGROUND Multi-frequency bioelectrical impedance analysis (MF-BIA) has been used to evaluate extracellular fluid volume (ECFV), but not fluid fluxes associated with fluid or furosemide administration in horses. If able to detect acute changes in ECFV, MF-BIA would be useful in monitoring fluid therapy in horses. OBJECTIVE The purpose of this study was to evaluate the ability of MF-BIA to detect acute fluid compartment changes in horses. We hypothesized that MF-BIA would detect clinically relevant (10-20%) changes in ECFV. METHODS Six healthy mares were used in the study. METHODS This is an original experimental study. Mares were studied in 3 experiments: (1) crystalloid expansion of normally hydrated subjects, (2) furosemide-induced dehydration followed by crystalloid administration, and (3) acute blood loss followed by readministration of lost blood. MF-BIA measurements were made before, during, and after each fluid shift and compared to known changes in volume calculated based on the intravenous fluids that were administered in addition to urinary fluid losses. Mean errors between MF-BIA estimated change and known volume change were compared using nonparametric analysis of variance. Estimated ECFV pre- and post-fluid administration similarly were compared. The level of statistical significance was set at P < .05. RESULTS Results of the study revealed a statistically significant change in ECFV and total body water during crystalloid expansion and dehydration. Statistically significant changes were not observed during blood loss and administration. Mean errors between MF-BIA results and measured net changes were small. CONCLUSIONS MF-BIA represents a practical and accurate means of assessing acute fluid changes during dehydration and expansion of ECFV using isotonic crystalloids with potential clinical applications in equine critical care.