The heparin dose-response (HDR) technique is based on activated clotting time (ACT) response to a fixed-dose heparin bolus, which varies substantially among patients. It is unclear, however, whether hemodilution-associated reductions in coagulation and anticoagulation factors affect the HDR slope. For in vitro hemodilution, aliquots of whole blood from healthy volunteers were diluted 9:1 and 8:2 v/v with normal saline. For in vivo hemodilution, a prospective observational study was performed on 46 patients who underwent elective cardiovascular surgery with or without cardiopulmonary bypass. HDR slope, antithrombin (AT) activity, complete blood count, and other coagulation parameters were compared after induction of anesthesia and after hemodilution with 500ml of intravenous fluid. In vitro 10 and 20% hemodilution significantly increased the HDR slope relative to baseline, reducing the heparin requirement. Hemodilution of heparinized samples significantly prolonged ACT, whereas there was no significant change in non-heparinized blood. The percent changes in fibrinogen and AT activity were significantly greater at 20% than those of the other coagulation variables. In vivo, hemodilution significantly increased the HDR slope and reduced heparin requirement. The percent change in fibrinogen due to hemodilution was significantly greater than the change in AT activity. Target ACTs of 300 and 450s were not achieved in 83.3 and 53.8% of patients, respectively. In vitro and in vivo hemodilution significantly increased the HDR slope and reduced the requirement for heparin. In vitro, the HDR slope did not change in parallel but became steeper, depending on the degree of hemodilution.