Topical glyceryl trinitrate treatment has demonstrated short- to mid-term efficacy in chronic noninsertional Achilles tendinopathy. However, the underlying mechanisms are far from being understood. We hypothetized that Achilles tendon capillary blood flow changes immediately after topical glyceryl trinitrate treatment. Fifteen patients (55 ± 15 years, VAS 5.8 ± 2.3) with current mid-portion Achilles tendon pain 36 months after open surgical Achilles tendon repair for tendon rupture were included. On the Achilles mid-portion, 1.2 mg topical glyceryl trinitrate was sprayed. Microcirculatory monitoring included capillary blood flow, tendon oxygen saturation and postcapillary venous filling pressures at the insertion and 2, 4 and 6 cm above the insertion using a combined laser Doppler and spectrophotometry system. Baseline capillary blood-flows of the painful versus the uninjured tendon were increased [108 ± 46 vs. 81 ± 20 (2 cm above the insertion), 104 ± 40 vs. 76 ± 20 (4 cm above the insertion), 111 ± 53 vs. 90 ± 21 (6 cm above the insertion, P < 0.05)]. However, topical glyceryl trinitrate did not change capillary blood-flow at 2 and 8-mm tissue depths at the painful Achilles tendon or the healthy tendon. Tendon oxygenation was not changed at the painful or the healthy Achilles tendon. Postcapillary venous filling pressure was reduced at 8 mm at the mid-portion in the painful Achilles tendon only (113 ± 37 vs. 95 ± 31, P = 0.030). Acute topical glyceryl trinitrate facilitates capillary venous outflow in painful Achilles tendons. However, capillary blood-flow and tendon oxygenation remain unchanged following acute topical glyceryl trinitrate application. Elevated capillary blood-flow at the entire mid-portion is encountered at baseline in previously ruptured painful Achilles tendons even 3 years after surgical repair of the Achilles tendon indicating an altered microcirculatory flow pattern.