An important factor in quality control of non-invasive prenatal screening (NIPS) or testing (NIPT) is a sufficient percentage of fetal DNA to avoid false-negative results. Here we evaluate 14,379 shallow whole-genome sequenced diagnostic NIPS samples, as well as negative controls, for both technical and biological factors that can influence fetal fraction and its assessment. Technically, bioinformatics analyses can have a profound impact on fetal fraction determination. We found best performance for fetal fraction determination with the Y chromosome based tool DEFRAG for male fetuses and the count based tool SeqFF for female fetuses. Biologically, gestational age of up to 21 weeks and maternal age had no influence on fetal fraction, while an increase in weight and BMI had a negative influence on fetal fraction. While a trend was observed, no statistically significant difference in fetal fraction was found between trisomy and normal samples. Overall, these results confirm the influence of biological factors and give insight into technical factors that can affect fetal fractions in NIPS.