Abstract Introduction The main generator of delayed onset muscle soreness (DOMS) is still unknown. This study aimed to clarify the main generator of DOMS. Methods Twelve participants performed eccentric exercise (EE) on lower legs. MRI and ultrasound were used to assess changes of calf muscle and deep fascia before and after EE. These results were then compared to the muscle pain level. Results Compared to baseline, muscle pain peaked at 24–48 h after EE (downstairs 22.25 ± 6.196, 57.917 ± 9.298, F = 291.168, p