Objective: Neuronal-specific enolase (NSE) and protein S100B have gained considerable interest as the markers of CNS injury, glial cell activation, and/or blood-brain barrier (BBB) disruption. No studies have investigated NSE and S100B in cluster headache (CH), but these biomarkers could contribute to the understanding of CH. Methods: Patients with episodic CH in bout (eCHa), in remission (eCHr), and chronic CH (cCH) were included in this randomized, double-blind, placebo-controlled, 2-way cross-over provocation study carried out at the Danish Headache Center. The primary endpoints included (1) differences of NSE and S100B in between groups (eCHa, eCHr, and cCH) at baseline; (2) differences over time in plasma concentrations of NSE and S100B between patient developing an attack and those who did not; (3) differences in plasma concentrations over time of NSE and S100B between active day and placebo day. Baseline findings were compared to the historical data on migraine patients and healthy controls and presented with means ± SD. Results: Nine eCHa, 9 eCHr, and 13 cCH patients completed the study and blood samples from 11 CGRP-induced CH attacks were obtained. There were no differences in NSE levels between CH groups at baseline, but CH patients in active disease phase had higher levels compared with 32 migraine patients (9.1 ± 2.2 µg/L vs 6.0 ± 2.2 µg/L, P <.0001) and 6 healthy controls (9.1 ± 2.2 µg/L vs 7.3 ± 2.0 µg/L, P =.007). CGRP-infusion caused no NSE changes and, but a slight, non-significant, increase in NSE was seen in patients who reported a CGRP-induced CH attack (2.39 µg/L, 95% Cl [−0.26, 3.85], P =.061). At baseline S100B levels in eCHa patients were higher compared to cCH patients (0.06 ± 0.02 µg/L vs 0.04 ± 0.02 µg/L, P =.018). Infusion of CGRP and CGRP-induced attacks did not change S100B levels. Apart from induced CH-attacks no other adverse events were noted. Conclusions: At baseline eCHa patients had higher S100B plasma levels than cCH patients and there was a slight, however not significant, NSE increase in response to CGRP-induced CH attack. Our findings suggest a possible role of an ictal activation of glial cells in CH pathophysiology, but further studies are warranted.