Human milk proteins are known as vital molecules for infant development and growth. Tissue factor is one of these human milk proteins that its role in human milk has not been cleared yet. Therefore, the first aim of this study was to detect the tissue factor activity of human milk and also was to investigate the effect of extended freezer storage on the milk tissue factor activity. The relationship between the tissue factor activity and macronutrient content and pH of milk was also investigated in this study. Under this aim, mature human milk samples were obtained from 8 healthy women. Collected human milk samples were pooled and divided into aliquots that were stored at - 20 degrees C until the day to be analyzed. Milk tissue factor activity, protein, fat, lactose, energy, water, density, and pH levels were determined for up to six months. By two months from the freezing, tissue factor activity did not significantly change but significantly decreased at the end of the six months. From the first month to six months from freezing, lactose, protein, fat, and energy levels showed a significant decline. Milk pH did not change with freezing at the end of 6 months. In conclusion, TF activity maintained its first-day activity until the second month after being pumped. The increased interest in breast milk leads us to believe that the gap existing in the knowledge of breast milk bioactive components like TF will be complemented with new research data.