Neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP) have emerged as biomarkers for cerebral small vessel disease (SVD). We investigated their role in a hereditary SVD model, retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL-S). NfL and GFAP levels of 17 pre-symptomatic, 22 symptomatic RVCL-S mutation carriers and 69 controls were measured using a Simoa assay. We assessed the association of serum and cerebrospinal fluid (CSF) levels of NfL and GFAP with RVCL-S symptomatology and neuropsychological functioning. Serum and CSF NfL levels were higher in symptomatic RVCL-S compared to controls ≥ 45years (33.5pg/mL vs. 9.2pg/mL, p < 0.01; 8.5*102pg/mL vs. 3.9*102pg/mL, p < 0.01, respectively). Serum NfL levels were higher in symptomatic RVCL-S than pre-symptomatic carriers (33.5pg/mL vs. 5.9pg/mL, p = 0.02). Pre-symptomatic RVCL-S carriers had increased CSF NfL levels compared to controls < 45years (5.2*102pg/mL vs. 1.9*102pg/mL, p < 0.01). No differences were found in GFAP levels across groups, but in RVCL-S carriers higher serum levels of both NfL and GFAP were linked to poorer global cognitive functioning (β[95%CI] = -2.86 [-5.58 to -0.13], p = 0.04 and β[95%CI] = -6.85 [-11.54 to -2.15], p = 0.01, respectively) and prolonged psychomotor test times (β[95%CI] = 6.71 [0.78-12.65], p = 0.03 and β[95%CI] = 13.84 [3.09-24.60], p = 0.01). Higher levels of serum NfL and GFAP are associated with worse cognitive functioning in RVCL-S carriers and may serve as marker for disease progression. CSF NfL levels may serve as early marker as pre-symptomatic RVCL-S patients already show differences compared to young controls.