OBJECTIVE: Between 50% and 67% of adults suffer from neck-shoulder pain, which may be associated with increased stiffness of neck muscles. We assessed pressure pain sensitivity and muscle stiffness maps of the upper trapezius in female computer users with and without chronic neck-shoulder pain and investigated the effects of eccentric training on females with neck-shoulder pain. DESIGN: Cross-sectional (part 1) and open-label (part 2) study. SETTING: University. SUBJECTS: Twenty females with neck-shoulder pain were compared with 20 controls (part 1). In part 2, neck-shoulder pain participants followed a five-week unilateral upper trapezius eccentric training program. METHODS: Topographical maps of pressure pain thresholds (pressure algometer) and muscle stiffness (myotonometer), using a 15-point grid covering myotendinous and muscle belly sites, and shoulder elevation force and range of elevation (dynamometer) were assessed at baseline and after training. RESULTS: There were no differences in pressure pain thresholds between sites (P = 0.243) or groups (P = 0.068), and there were significant differences in stiffness between myotendinous and muscle belly sites (P < 0.001) but not groups (P = 0.273). After training, pressure pain thresholds increased, stiffness decreased (P < 0.005), and shoulder elevation force and range of elevation improved (P < 0.001). CONCLUSIONS: The lack of differences in upper trapezius pressure pain sensitivity and stiffness between females with or without neck-shoulder pain confirms no clear etiology among computer users reporting neck-shoulder pain. A five-week eccentric training protocol showed positive effects on pressure pain sensitivity, stiffness, shoulder force, and range of motion.