Thrombin generation measurement is becoming a useful technique to assess the hemostatic system. Subsampling methods though labour intensive, measure activities directly. The continuous method has become an alternative option. Studies by Hemker et.al. (1) showed that both methods gave comparable results but had some differences. We decided to further assess the variation between the methods. For the continuous method, 125μl of plasma, 15μl of Pefabloc (50mg/ml) and 12.5μl of Pefachrome TG5134 (0.5mM) were added to a 96 well ELISA plate and prewarmed to 37°C. The reaction was started by adding 97.5μl of 1/12.5 Thromborel S in 25.64mM CaCl2/Tris buffer. The optical density at 405nm was recorded at an interval of 15sec for 15 min. For the subsampling method, 125μl of 1/16 Thromborel S in 20mM CaCl2/Tris buffer was added to 125μl of defibrinated (Arvin) plasma. Subsamples were taken into 5mM EDTA solution at 15sec intervals. Chromogenix S2238 was used for measuring thrombin in the subsampling method. The continuous method gave a peak height of ~410nM and area under the curve of 860–900 nM.min whereas the subsampling method gave a peak height of ~360nM and an area under the curve of 550–600nM.min. This difference disappeared when PefachromeTG5134 was added into the reaction mixture prior to running subsampling assays. Thus, substrate likely slowed inhibition of generated thrombin in the continuous assay, which confirms previous findings by Hemker et.al. (1). Further examination revealed that the concentration of α2M-IIa complex was very different between the two methods. α2M-IIa complex calculated from the continuous method (80nM) was much greater than α2M-IIa complex obtained experimentally by the subsampling method (38nM) even in the presence of TG5134. However, addition of a large excess of antithrombin and heparin at the final equilibrium time point of the continuous assay reduced the α2M-IIa complex to the same level (38nM) as that of the subsampling method. We have shown that the slow-acting substrate Pefachrome TG5134 gave an increased estimate of both free and α2M-IIa complex. Thus, a significant amount of free thrombin remains at the end of thrombin generation that is not accounted for in the continuous method. This estimate of final α2M-IIa complex concentration may be important in determining the relative protection against long-term plasma thrombin activity in pediatric patients with elevated α2M levels.