Ten healthy non-smoking males (mean age 24.3+/-0.8 years) with no history of chronic muscle pain or migraine participated in this study. Intramuscular total haemoglobin (Hb), an indicator of blood volume in the illuminated area, was measured with a non-invasive, near-infrared spectroscopic device. Each participant was told to maintain maximal mouth opening to extend the masseter muscle for 30, 60 or 120 s in random order. Data were continuously recorded from the right masseter 1 min before, at set times during and for 5 min after sustained muscle extension in each trial. Each trial was separated by a 10-min interval. Heart rate (HR) and blood pressure (BP) were also recorded. The mean normalized Hb decreased during muscle extension and rebound hyperaemia was observed after it in each trial (P=0.0001). Hb returned to baseline within 60 s. The magnitude of the decremental change during extension and of the incremental change in the rebound hyperaemia was not significantly different among the three trials (P=0.9071); neither were mean normalized HR and BP. These data suggest that sustained extension of the masseter produces a reduction in total intramuscular Hb during extension and a secondary increase in Hb following a return to the resting muscle's normal length.