Background Although accumulating evidence suggests that the hemostatic balance is impaired in patients with hypertriglyceridemia, hyperbilirubinemia or hemolytic anemias, little is known on the underlying biological mechanisms. This experimental study was aimed at exploring whether increasing values of triglycerides, bilirubin or cell-free hemoglobin promote thrombin generation in plasma. Methods Three different pools were prepared from three different sets of 20 normal routine plasma citrate samples. The native pools were spiked with increasing amounts of exogenous triglycerides (up to 8.8 mmol/L), bilirubin (up to 350 μmol/L) or autologous hemolyzed blood (up to 3.5 g/L cell-free hemoglobin). Using the fully-automated thrombin generation analyzer ST Genesia, we measured the following parameters: lag time (LT), time to peak (TP), peak height (PH) and endogenous thrombin potential (ETP). Results A sustained increase of PH and ETP was found in parallel with increasing triglyceride concentrations, peaking in the aliquot with 8.8 mmol/L. Conversely, LT and TP displayed an opposite trend, reaching a maximum decrease in the 8.8 mmol/L aliquot. Increasing bilirubin concentrations promoted remarkable increases of PH and ETP and decreases of TP and LT, up to 211 μmol/L. After this threshold, all parameters tended to return towards baseline values. A constant increase of PH and ETP was also noted in hemolyzed samples, peaking in the 3.5 g/L cell-free hemoglobin aliquot, whereas the TP and LT remained unchanged in all hemolyzed aliquots. Conclusions Our findings suggest that hypertriglyceridemia, hyperbilirubinemia and hemolysis may promote a hypercoagulable state in human plasma.