Abstract Background: Trisomy 21 risk estimation in first trimester pregnancies can be performed by a combined test based on ultrasound measurement of fetal nuchal translucency thickness and maternal plasma concentrations of free ß human chorionic gonadotropin (hCGß) and pregnancy-associated plasma protein A (PAPP-A). However, little knowledge exists regarding the biological variation of hCGß and PAPP-A when the time interval between sampling increases. Methods: We estimated these variations from double mea-surements of hCGß and PAPP-A in first trimester pregnancies in 167 women. Data were divided into three groups based on the number of days between sampling. The correlation coefficients and biological variation were estimated for each group. Results: The correlation coefficient between the first and second samples was 0.841 for hCGß, and 0.706 for PAPP-A. The ranges for biological variation were 11.9%-48.5% for hCGß and 31.6%-63.3% for PAPP-A, increasing with time between sampling. The average overall biological variation for hCGß was 29%, and 49.7% for PAPP-A. Conclusions: We found high biological variation for plasma concentrations of hCGß and PAPP-A, increasing with longer time intervals between sampling. From our data that showed high correlation of hCGß and PAPP-A in the first and second sample, we found no reason to recommend retesting. However, new studies should clarify whether PAPP-A should be collected early, and hCGß late, in the first trimester of pregnancy.