Background: Prompt intervention can prevent permanent adverse neurological effects caused by neonatal hypothyroidism. Thyroid function changes rapidly in the first few days of life but well-defined age-specific reference intervals (RIs) for thyroid-stimulating hormone (TSH), free thyroxine (FT4) and free tri-iodothyronine (FT3) are not available to aid interpretation. We developed hour-based RIs using data mining. Methods: All TSH, FT4 and FT3 results with date and time of collection from neonates aged <7 days during 2005–2015 were extracted from the Monash Pathology database. Neonates with more than one episode of testing or with known primary hypothyroidism, identified by treating physicians or from medical records, were excluded from the analysis. The date and time of birth were obtained from the medical records. Results: Of the 728 neonates qualifying for the study, 569 had time of birth available. All 569 had TSH, 415 had FT4 and 146 had FT3 results. For age ≤24 h, 25–48 h, 49–72 h, 73–96 h, 97–120 h, 121–144 h and 145–168 h of life, the TSH RIs (2.5th–97.5th) (mIU/L) were 4.1–40.2, 3.2–29.6, 2.6–17.3, 2.2–14.7, 1.8–14.2, 1.4–12.7 and 1.0–8.3, respectively; the FT4 RIs (mean ± 2 standard deviation [SD]) (pmol/L) were 15.3–43.6, 14.7–53.2, 16.5–45.5, 17.8–39.4, 15.3–32.1, 14.5–32.6 and 13.9–30.9, respectively; the FT3 RIs (mean±2 SD) (pmol/L) were 5.0–9.4, 4.1–9.1, 2.8–7.8, 2.9–7.8, 3.5–7.2, 3.4–8.0 and 3.8–7.9, respectively. Conclusions: TSH and FT4 were substantially high in the first 24 h after birth followed by a rapid decline over the subsequent 168 h. Use of hour-based RIs in newborns allows for more accurate identification of neonates who are at risk of hypothyroidism.