The association of hyperhomocysteinemia and hypercysteinemia with the risk of arterial and venous thrombosis is well documented. While it is known that standardized pre-analytical conditions are necessary for reliable measurement of plasma total homocysteine, the effects of pre-analytical conditions on cysteine measurement are less well known. The aim of this study was to evaluate the effects of pre-analytical conditions on the measurement of homocysteine and cysteine. We observed that the concentration of total homocysteine in plasma increased significantly with time (38% after 6 h), whereas total cysteine decreased (5% after 2 h) when blood anticoagulated with ethylenediaminetetraacetic tripotassium salt was kept at room temperature. These changes were minimized when acidic citrate dextrose was used as an anticoagulant and were abolished when blood samples were immediately placed on crushed ice, independently of the anticoagulant. Storage of plasma for 72 h at room temperature induced a small (approximately equal to 6%), but significant, decrease in cysteine when blood was collected in ethylenediaminetetraacetic tripotassium salt. In contrast, homocysteine was stable in plasma for 72 h, independently of the anticoagulant used. In conclusion, if blood samples for plasma total homocysteine and cysteine measurement cannot be kept on ice, they should be collected in acidic citrate dextrose to minimize the artifactual changes.