Summary Aim: The aim of this study was to establish normal values for thyroid uptake of 99mtechnetium pertechnetate (99mTcO4) as a function of age, sex and circadian rhythm in mice. Methods: In 12 female (F) and 12 male (M) C57BL/6N mice, nine consecutive SPECT images of 10 min duration each were acquired as dynamic acquisitions beginning 5 min after intravenous injection of 80 MBq 99mTcO4. Each mouse was imaged in follow-up studies up to 24 months (A: 1 month; B: 3 months; C: 6 months; D: 12 months; E: 24 months). In order to assess for physiologic changes related to circadian rhythm, animals were imaged during light (sleeping phase, SP) as well as during night conditions (awake phase, AP). The percentage tracer uptake of the injected activity is expressed as median %ID. Results: Female mice showed significantly higher uptake than males (F 1.6, M 1.1; p < 0.001). This effect was observed up to the age of 12 months: A (F 1.6, M 1.1; p < 0.001), B (F 1.7, M 1.1; p < 0.001), C (F 1.8, M 1.2; p < 0.001), D (F 1.6, M 1.2; p < 0.001), E (F 1.1, M 1.1; p = 0.79). Impact of age on uptake could be observed in females (p = 0.056) and was not present in males (p = 0.27). A significant effect of circadian rhythm could not be observed in females (SP 1.6, AP 1.7; p = 0.65) but in males (SP 1.2, AP 1.1; p = 0.02). Conclusion: This study showed a significant influence of sex on thyroid 99mTcO4 uptake in mice. Sex was also a significant factor affecting age-related changes in uptake in female mice but not in males. In contrast, circadian rhythm had no relevant impact on 99mTcO4 uptake. Therefore, design of thyroid uptake studies in mice using 99mTcO4 should consider animal sex, and in females, age as significant factors affecting uptake.