Summary. Diagnostic tests play an essential role in childrens health. Previous work has shown variation in the use of diagnostic tests for adults. However, comparatively little is known about the use of tests in children. We aimed to analyze temporal trends in laboratory testing for children aged 0 to 15 from 2005 to 2019 in Oxfordshire, United Kingdom. Methods. For this retrospective analysis, we used data from the Oxfordshire University Hospital NHS Trust laboratories. Using joinpoint regression models, we estimated annual percentage changes (APC) in test use. Temporal changes in age-adjusted rates in test use were calculated overall and stratified by healthcare setting, sex and age. Findings. Between 2005 and 2019, overall test use increased in children (APC 1.6%, 95 confidence interval -0.8% to 4.1%). Increases were highest in females, in those aged 11-15 years and the outpatient setting. The most frequently requested tests were full blood count, urea and electrolytes, liver function test, C-reactive protein and calcium magnesium phosphate. The test with the greatest increase in use was Vitamin D, which increased on average by 27% per year. Other tests that showed a significant temporal increase included parathyroid hormone, iron studies, folate, vitamin B12 levels, glucose, HbA1c, IgA, coeliac, creatine kinase, thyroid function tests and IgG/IgM. Test changes were not uniformly distributed across all settings and age groups. Interpretation. The increase in test use may be the result of a combination of factors, including changes to the health service resulting in an increased volume of presentations and referrals, shifts in workforce composition towards less experienced clinicians, increased parental anxiety and expectation of tests and/or increased awareness and changing prevalence of disease. Further research is needed to quantify whether test use is warranted and to compare trends in Oxfordshire with other settings. Funding. No funding was obtained for this study.