This prospective study examined the value of serum neurofilament protein levels for detecting peri-operative brain damage following carotid endarterectomy. An ELISA was used for quantification of neurofilament protein heavy chain (NfHSMI35) levels from patients undergoing endarterectomy for symptomatic (n = 17) and asymptomatic high-grade internal carotid artery stenosis (n = 30). All patients underwent diffusion-weighted brain imaging before and after the procedure. NfHSMI35 levels were significantly higher in patients with a symptomatic carotid artery stenosis (0.131 ng/ml) if compared to asymptomatic patients (0.055 ng/ml, P = 0.01). However, serum NfHSMI35 levels were not related to signs of brain ischemia on routine brain imaging techniques. Our pilot data suggests that raised NfHSMI35 serum levels in patients with symptomatic carotid artery disease may be a sensitive biomarker for diffuse ischemic damage to the CNS. We conclude that NfHSMI35 failed to qualify as a biomarker for peri-operative brain injury in CEA and factors that may have compromised the validation of this biomarker are discussed and need to be taken into account for the design of further studies.