Several chronic pain conditions are accompanied with enlarged referred pain areas. This study investigated a novel method for assessing referred pain. In 20 healthy subjects, pressure pain thresholds (PPTs) were recorded and pressure stimuli (120% PPT) were applied bilaterally for 5 and 60 seconds at the infraspinatus muscle to induce local and referred pain. Moreover, PPTs were measured bilaterally at the shoulder, neck, and leg before, during, and after hypertonic saline–induced referred pain in the dominant infraspinatus muscle. The pressure and saline-induced pain areas were assessed on drawings. Subsequently, delayed onset muscle soreness was induced using eccentric exercise of the dominant infraspinatus muscle. The day-1 assessments were repeated the following day (day 2). Suprathreshold pressure stimulations and saline injections into the infraspinatus muscle caused referred pain to the frontal aspect of the shoulder/arm in all subjects. The 60-second pressure stimulation caused larger referred pain areas compared with the 5-second stimulation (P < 0.01). Compared with pressure stimulation, the saline-induced referred pain area was larger (P < 0.02). After saline-induced pain, the PPTs at the infraspinatus and supraspinatus muscles were reduced (P < 0.05), and the 5-second pressure-induced referred pain area was larger than baseline. Pressure pain thresholds at the infraspinatus and supraspinatus muscles were reduced at day 2 in the delayed onset muscle soreness side (P < 0.05). Compared with day 1, larger pressure and saline-induced referred pain areas were observed on day 2 (P < 0.05). Referred pain to the shoulder/arm was consistently induced and enlarged after 1 day of muscle soreness, indicating that the referred pain area may be a sensitive biomarker for sensitization of the pain system.