Background: Plasma biomarkers may be useful to detect healthy individuals at increased risk for atherosclerotic manifestations, such as carotid artery stenosis. The aim of this longitudinal cohort study was to evaluate new biomarkers in relation to C-reactive protein and conventional risk factors for carotid artery stenosis during long term follow-up Methods: The following markers were measured in 5550 middle-aged subjects: C-reactive protein, lipoprotein-associated phospholipase A2, proneurotensin, midregional pro-adrenomedullin, midregional pro-atrial natriuretic peptide, N-terminal pro B-type natriuretic peptide, copeptin, and cystatin C. Subjects with prevalent carotid artery stenosis were excluded. Subjects were followed in national patient registers for 23.4 (interquartile range 19.5À24.3) years regarding incident carotid artery stenosis, both operated and non-operated. Results: When including conventional risk markers in Cox regression, N-terminal pro B-type natriuretic peptide (Hazard ratio 1.36; 95% confidence interval 1.12À1.65; p = 0.002) was independently associated with incident carotid artery stenosis, whereas there were trends for C-reactive protein (HR 1.20; 95% confidence interval 0.98À1.48; p = 0.071), and midregional pro-adrenomedullin (Hazard ratio 1.21; 95% confidence interval 0.99À1.47; p = 0.061). Midregional pro-adrenomedullin (Hazard ratio 1.30; 95% confidence interval 1.03À1.65; p = 0.029) was independently associated with incident surgery for carotid artery stenosis, whereas there was a trend for N-terminal pro B-type natriuretic peptide (Hazard ratio 1.31; 95% confidence interval 1.00À1.72; p = 0.052). Conclusions: N-terminal pro B-type natriuretic peptide and midregional pro-adrenomedullin can be used as predictors for clinically detected carotid artery stenosis during long-term follow-up of healthy subjects.