Although reflexes in human jaw muscles have been extensively studied, the neurotransmitters involved in the regulation of these reflexes are not well known. The aim of the present study was to investigate whether amitriptyline, a combined serotonin and noradrenaline re-uptake inhibitor, modulates the late exteroceptive suppression period (ES2) of temporal muscle activity in chronic tension-type headache. ES2 was recorded with a previously evaluated method and assessed by a blinded observer in 35 patients with chronic tension-type headache. Thereafter, ES2 was recorded in 27 of these patients during a double-blind, placebo-controlled, 3-way crossover trial investigating the prophylactic effect of amitriptyline, the selective serotonin re-uptake inhibitor citalopram, and placebo. ES2 duration was significantly shorter during treatment with amitriptyline than during placebo, P = 0.02, while ES2 duration only tended to be shorter during treatment with citalopram, P = 0.34. ES2 was not significantly correlated to the prophylactic effect of amitriptyline or to a range of clinical and experimental pain parameters. Our results demonstrate that amitriptyline reduces ES2 and indicate that ES2 is modulated by serotonergic as well as noradrenergic neuronal pathways.