Objective Neurofilaments are key axonal proteins, with neurofilament light (NfL) and heavy (NfH) chain recognized as promising biomarkers for neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, neurofilament medium chain (NfM) remained previously underexplored due to a lack of quantitative assays. In this study, we developed a sensitive immunoassay to measure NfM in cerebrospinal fluid (CSF) and analyzed its levels in ALS, Alzheimer's disease (AD), frontotemporal dementia (FTD), and Lewy body dementia (LBD). Correlations among neurofilaments and their diagnostic performance were also evaluated. Methods In this study CSF levels of three neurofilament proteins were measured in 271 participants, including patients with ALS (n=91), AD (n=25), FTD (n=38), LBD (n=18), non-neurodegenerative controls (CTRL, n=51), and 48 individuals initially evaluated for ALS but ultimately diagnosed with other conditions (CTRL.DD). Results All three neurofilaments were significantly elevated in ALS compared to CTRL and CTRL.DD groups (p<0.0001 for both), with NfM and NfL also increased in FTD (p<0.0001 for both) and AD (NfM, p=0.0017; NfL, p=0.0135 ) compared to CTRL. NfH demonstrated the greatest distinction between ALS and FTD (p<0.0001). Strong correlations were observed among neurofilament subunits, particularly between NfM and NfL (r=0.94, 95% CI: 0.93-0.96, p<0.0001). All neurofilaments effectively distinguished ALS from CTRL and CTRL.DD, with AUC values ranging from 0.92 to 0.99. NfM and NfL showed high accuracy in differentiating AD (NfM, AUC: 0.89; NfL, AUC: 0.90) and FTD (NfM, AUC: 0.91; NfL, AUC: 0.92) from CTRL, while NfH best separated ALS from FTD (AUC: 0.96). Conclusion This study provides the first quantitative comparison of NfM with NfL and NfH in a neurodegenerative cohort, highlighting its potential diagnostic value. Further research with larger cohorts, longitudinal studies, and investigations into neurofilament distribution in different compartments is needed to clarify the distinct roles of NfM, NfL, and NfH in the diagnosis and treatment of neurological diseases.

HT reports honoraria for acting as a consultant/speaker and/or for attending events sponsored by Alexion, Bayer, Biogen, Bristol-Myers Squibb, Celgene, Diamed, Fresenius, Fujirebio, GlaxoSmithKline, Horizon, Janssen-Cilag, Merck, Novartis, Roche, Sanofi-Genzyme, Siemens, Teva and Viatris. All conflicts are not relevant to the topic of the study. All other authors declare no competing interests.

The present study was supported by the Else Kroener Fresenius Foundation and Chemische Fabrik Karl Bucher GmbH.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The study was approved by the Ethics Committee of the University of Ulm (approval number: 20/10) and was conducted in accordance with the Declaration of Helsinki.

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All data produced in the present study are available upon reasonable request to the corresponding author.