The objective of this study was to verify the effects of aerobic exercise associated with tryptophan (TRP) supplementation on hyperalgesia, as well as on cortisol, IL-6 and TNF concentrations in female rats with experimental fibromyalgia (FM). Female Wistar rats (initial body weight: ~ 350 g; age: 12 months) were randomly divided into 5 groups: CON (Control); F (Fibromyalgia induced); FE (Fibromyalgia induced plus exercise); FES (Fibromyalgia induced plus exercise and TRP supplementation) and FS (Fibromyalgia induced plus TRP supplementation). Fibromyalgia was induced with two injections (20 μL) of acidic saline (pH 4.0) into the right gastrocnemius muscle with a 3-day interval. Control animals received the same doses of neutral saline (pH 7.4). The exercised animals underwent progressive low-intensity aerobic exercise (LIAE) on a treadmill (10–12 m/min, 30–45 min/day, 5 days/week) for three weeks. During this period, the supplemented animals received a TRP supplemented diet (210 g/week), while the others received a control diet. Mechanical hyperalgesia was evaluated weekly and serum cortisol and muscle IL-6 and TNF concentrations were assessed after three weeks of interventions. Experimental FM caused bilateral hind paw hyperalgesia and augmented serum cortisol and muscle IL-6 concentrations. After 3 weeks of interventions, LIAE alone reduced hyperalgesia (151%) and reduced serum cortisol concentrations (72%). Tryptophan supplementation itself diminished hyperalgesia (57%) and reduced serum cortisol concentrations (67%). Adding TRP supplementation to LIAE did not further reduce hyperalgesia significantly (11%), which was followed by an important decrease in muscle IL-6 concentrations (68%), though reduction in serum cortisol pulled back to 45%. Muscle TNF concentrations were not affected. In conclusion, the association of TRP supplementation to LIAE does not potentiate significantly the reduction of bilateral mechanical hyperalgesia promoted by LIAE in female rats with experimental FM, however an important decrease in IL-6 is evident.