PURPOSE:White matter fibre development in childhood involves dynamic changes to microstructural organisation driven by increasing axon diameter, density, and myelination. However, there is a lack of longitudinal studies that have quantified advanced diffusion metrics to identify regions of accelerated fibre maturation, particularly across the early pubertal period. We applied a novel longitudinal fixel-based analysis (FBA) framework, in order to estimate microscopic and macroscopic white matter changes over time. METHODS:Diffusion-weighted imaging (DWI) data were acquired for 59 typically developing children (27 female) aged 9-13 years at two time-points approximately 16 months apart (time-point 1: 10.4 ± 0.4 years, time-point 2: 11.7 ± 0.5 years). Whole brain FBA was performed using the connectivity-based fixel enhancement method, to assess longitudinal changes in fibre microscopic density and macroscopic morphological measures, and how these changes are related to sex, pubertal stage, and pubertal progression. Follow-up analyses were performed in sub-regions of the corpus callosum to confirm the main findings using a Bayesian repeated measures approach. RESULTS:There was a statistically significant increase in fibre density over time localised to medial and posterior commissural and association fibres, including the forceps major and bilateral superior longitudinal fasciculus. Increases in fibre cross-section were substantially more widespread. The rate of fibre development was not associated with age or sex. In addition, there was no significant relationship between pubertal stage or progression and longitudinal fibre development over time. Follow-up Bayesian analyses were performed to confirm the findings, which supported the null effect of the longitudinal pubertal comparison. CONCLUSION:Using a novel longitudinal fixel-based analysis framework, we demonstrate that white matter fibre density and fibre cross-section increased within a 16-month scan rescan period in specific regions. The observed increases might reflect increasing axonal diameter or axon count. Pubertal stage or progression did not influence the rate of fibre development in the early stages of puberty. Future work should focus on quantifying these measures across a wider age range to capture the full spectrum of fibre development across the pubertal period.