The aim of our present study was to establish new reference intervals for thyrotropin (TSH) and thyroid hormones based on National Academy of Clinical Biochemistry (NACB) criteria and regular thyroid ultrasonography. We also assessed the effect of potentially confounding factors to modulate the limits of these intervals. We investigated 870 apparently healthy persons and excluded, step by step, those with a family history of thyroid disease, pathologic thyroid ultrasonography results, and increased anti-thyroid peroxidase or anti-thyroglobulin antibodies. Accordingly, only 453 of the 870 persons in the entire group were finally included as reference collective. We measured serum concentrations of TSH, total and free thyroxine (T(4) and FT(4)), and total and free triiodothyronine (T(3) and FT(3)) of the whole and the reference collective on the ELECSYS system assays (Roche Diagnostics) and calculated the 2.5th and 97.5th percentiles for comparison. The calculated lower limit for TSH differed significantly between the reference intervals for healthy persons with an assessed normal thyroid gland vs the nonselected group of healthy blood donors. Age was the only independent factor and was significantly inversely associated with TSH (P <0.0001). Use of oral contraceptives was a significant predictor for variation in T(4) concentrations (P <0.001). Age and oral contraceptives were independently associated with T(3) variations (P <0.05). For FT(4) vs FT(3) variation, gender and (inversely) age (P <0.01) were independent modulating factors. The selection of healthy persons according to NACB criteria combined with sonographic confirmation of a normal thyroid gland provide a valid basis for the reference interval for TSH. Factors indicating a preclinical disease state, such as family history, pathologic ultrasonography result, or increased anti-thyroid peroxidase and anti-thyroglobulin antibodies, can be associated with normal hormone concentrations. Additionally, patient age and gender as well as use of contraceptives should be considered in diagnostic evaluation of thyroid diseases.