Adaptive alterations in maternal physiology cause changes in thyroid hormone levels throughout pregnancy, and precise biochemical evaluation is thus highly dependent on gestation-specific reference intervals and expected intra-individual variation. The aim of the study was the assessment of the intra-individual variation as well as the longitudinal course of thyroid hormones during normal pregnancy and factors that influence the normal reference range for thyroid function. For this purpose, a longitudinal statistical model was applied. In a cohort of 132 pregnant women, serial blood samples were obtained and ultrasound scans were performed throughout pregnancy. Serum levels of TSH, free and total thyroxine (T(4)), free and total triiodothyronine (T(3)) as well as autoantibodies against thyroid peroxidase and thyroglobulin were measured in 979 serum samples. Intra-individual variations of thyroid hormone concentrations were smaller than inter-individual variations (individuality index range: 0.38-0.71). Maternal height was positively associated with free T(4) (FT(4)) (b=0.003; P=0.031) and pre-pregnancy body mass index with T(3) and free T(3) (b=0.017; <0.001 and b=0.007; P<0.001). Smoking was positively associated with T(4) and FT(4), but it was modulated by gestational age. Gestation-specific reference intervals for thyroid function variables from autoantibody-negative participants are presented. In accordance with the data from nonpregnant adults, intra-individual variations of thyroid hormones were smaller than inter-individual variations also during pregnancy. In the evaluation of thyroid function in pregnancy, the individual longitudinal course of thyroid hormones rather than absolute values should be considered. We present a longitudinal model for the prediction of maternal thyroid function tests in pregnant women.