Objective: The aim of this work was to investigate the association between maternal and fetal characteristics and the fetal fraction at 8-14 weeks' gestation, with emphasis on the change in the fetal fraction upon repeat sampling. Method: One sample for cell-free DNA (cfDNA) testing was collected at the same time as the biochemical markers for combined first trimester screening (visit 1) and another at the nuchal translucency scan (visit 2). Chromosome-selective cfDNA analysis was performed on frozen plasma. Results: Overall, 321 women were included at visit 1, and 307 had a repeat blood sampling. A fetal fraction was obtained in 532 samples (238 samples with repeat fetal fraction). The fetal fraction decreased with maternal BMI (p < 0.001), was lower in Asian women (p = 0.03), and increased with β-hCG levels (p < 0.001) and gestational age (p = 0.04). Before 10 weeks' gestation, the fetal fraction was lower (p = 0.02), as was the probability of a sufficient fetal fraction (p = 0.03) after adjustment for maternal BMI. Asian women had a higher increase in fetal fraction upon repeat sampling (p < 0.001). Conclusion: Before 10 weeks' gestation, the fetal fraction is significantly lower but seems to increase more rapidly compared to later gestations. Presently, combined first trimester screening with cfDNA testing should not include samples before 10 weeks' gestation.