Abstract This study provides the first quantification of neurofilament light chain (NfL) kinetics in the human CNS using stable isotope labelling kinetics. NfL is elevated in CSF and blood across a range of traumatic, inflammatory and neurodegenerative diseases of the CNS, and has been increasingly included in clinical trials as a secondary or exploratory outcome measure of target engagement. Interpreting trajectories of NfL post-treatment has been challenging, prompting a greater need and focus on understanding its pathophysiology. We set out to measure NfL kinetics in the human CNS using stable isotope labelling kinetics. In human neurons derived from induced pluripotent stem cells, we show that NfL turnover is relatively slow, comparable to other long-lived proteins such as tau. We detected a delay of 3 to 6 days in the release of NfL into the media, unexpected for a passively released protein and supporting that controlled mechanisms of release could contribute to the appearance of NfL in the extracellular milieu. We optimized the kinetic NfL assay to measure the turnover of NfL in the human CNS. Participants with diagnosed primary tauopathies (n = 10) were recruited to the Human CNS Tau Kinetics in Tauopathies study and a cohort of cognitively unimpaired or with mild cognitive impairment (Clinical Dementia Rating score ≤0.5; n = 22) to the Tau Stable Isotope Labelling Kinetics study. Patients with suspected normal pressure hydrocephalus (n = 3) and primary tauopathy cases (n = 3) were examined to assess labelling in the brain parenchyma and ventricular CSF. In brain tissue, isotopically labelled in vivo and sampled ex-vivo and post-mortem, NfL is rapidly labelled but remains stable 18 months after, indicating very slow turnover and likely incorporation into very stable NfL networks. In line with a controlled mechanism of release in vivo, appearance of labelled NfL in CSF was detectable between 53 and 162 days post-labelling, during which NfL labelling did not reach its peak, suggestive of a half-life in CSF >3 months. These findings support the interpretation that acute rises in CSF NfL concentration are likely to be related to passive release or CSF clearance failure. We also show that active but delayed release of newly translated NfL can contribute to the concentration of NfL in CSF, but this would not be expected for at least 8 weeks. Clinical trials using NfL as an outcome measure will benefit from substantially longer follow-up periods and isotopic labelling to understand the NfL response to therapeutic intervention.