An emerging technique used for the study of metabolic regulation is the elevation of lactate concentration with a sodium-lactate infusion, the lactate clamp (LC). However, hematological and acid-base properties affected by the infusion of hypertonic solutions containing the osmotically active strong ions sodium (Na+) and lactate (Lac−) are a concern for clinical and research applications of LC. In the present study, we characterized the hematological and plasma acid-base changes during rest and prolonged, light- to moderate-intensity (55% V̇o2 peak) exercise with and without LC. During the control (Con) trial, subjects were administered an isotonic, isovolumetric saline infusion. During LC, plasma lactate concentration ([Lac−]) was elevated to 4 meq/l during rest and to 4–7 meq/l during exercise. During LC at rest, there were rapid and transient changes in plasma, erythrocyte, and blood volumes. LC resulted in decreased plasma [H+] (from 39.6 to 29.6 neq/l) at the end of exercise while plasma [HCO3−] increased from 26 to 32.9 meq/l. Increased plasma strong ion difference [SID], due to increased [Na+], was the primary contributor to decreased [H+] and increased [HCO3−]. A decrease in plasma total weak acid concentration also contributed to these changes, whereas Pco2 contributed little. The infusion of hypertonic LC caused only minor volume, acid-base, and CO2 storage responses. We conclude that an LC infusion is appropriate for studies of metabolic regulation.