We investigated the effect of chronic administration of different pain medications on the activity of the serotonin transporter (SERT) in patients with medication overuse headache (MOH). We measured the kinetic of platelet 5-HT uptake (maximal velocity, Vmax and the Michaelis-Menten constant, Km in patients with overuse of triptans (tMOH, n = 15) or analgesics (aMOH, n = 14) before and after drug withdrawal, as well as in headache-free healthy subjects (n = 15) and patients with episodic migraine (EM, n = 16). Vmax was increased similarly in both, tMOH and aMOH compared to healthy subjects and patients with EM and normalized after withdrawal in parallel to the improvement of headache frequency. Average Km was similar in all groups at baseline and not affected by the withdrawal. The data demonstrate a transient increase of SERT activity in patients with analgesic and triptan induced MOH but do not allow to differentiate whether the increase of serotonin uptake is caused by regular intake of analgesics or triptans or is a consequence of frequent headache attacks.