AbstractObjectiveThere is a need for sensitive biomarkers in amyotrophic lateral sclerosis (ALS), to enable earlier diagnosis and to help assess potential treatments. The main objective of this study was to compare two potential biomarkers, threshold‐tracking short‐interval cortical inhibition (T‐SICI), which has shown promise as a diagnostic aid, and neurofilament light chains (NfL).MethodsNinety‐seven patients with ALS (mean age 67.1 ± 11.5 years) and 53 ALS mimics (aged 62.4 ± 12.9) were included. Mean disease duration was 14 months ±14.1. Patients were evaluated with revised ALS functional rating score (ALSFRS‐R), Penn upper motor neuron score (UMNS), muscle strength using the Medical Research Council (MRC) score and examined with T‐SICI, quantitative electromyography (EMG), and NfL measured in spinal fluid.ResultsNfL increased with increasing UMNS (rho = 0.45, p = 8.2 × 10⁻⁶) whereas T‐SICI at 2.5 ms paradoxically increased toward normal values (rho = 0.53, p = 1.9 × 10⁻⁷). However, these two measures were uncorrelated. Discrimination between ALS patients and mimics was best for NfL (area under ROC curve 0.842, sensitivity 84.9%, specificity 83.5%), compared with T‐SICI (0.675, 39.6%, 91.8%). For the patients with no UMN signs, NfL also discriminated best (0.884, 89.3%, 82.6%), compared with T‐SICI (0.811, 71.4%, 82.6%). However, when combining NfL and T‐SICI, higher AUCs of 0.854 and 0.922 and specificities of 93.8 and 100 were found when considering all patients and patients with no UMN signs, respectively.InterpretationBoth T‐SICI and NfL correlated with UMN involvement and combined, they provided a strong discrimination between ALS patients and ALS mimics.