Delayed onset muscle soreness (DOMS) involves central and peripheral pain mechanisms. Referred pain patterns following stimulation of DOMS affected tissue have not been fully described. Referred pain may provide information on how central mechanisms are involved in DOMS, as referred pain is a central mechanism. Further, tendon tissue involvement in DOMS is not clear. This study assessed pressure pain threshold (PPT) sensitivity at the tendon, tendon-bone junction (TBJ) and muscle belly sites of tibialis anterior pre- and during DOMS in 45 subjects (34 males, 11 females). Furthermore, pain and referred pain areas at these three sites in response to hypertonic saline injection (n = 15 per injection site) were investigated pre- and during DOMS. DOMS was induced using controlled plantarflexion from a platform (bodyweight as resistance) causing eccentric contraction of the tibialis anterior muscle. DOMS induced PPT decrease was found at the TBJ and muscle belly sites only (P < 0.001). No mechanical effect was found in the unexercised limb. Maximal pain intensity induced by hypertonic saline given pre-DOMS was significantly higher for the tendon and TBJ injections compared to intramuscular injections (P < 0.05). Significantly higher referred pain frequency and enlarged pain areas were found at the muscle belly and TBJ sites following injection during DOMS compared to pre-DOMS. The results indicate that muscle belly and TBJ sites are sensitised while tendon tissue per se is unaffected by DOMS. Central sensitivity changes caused by DOMS may explain the increase in referred pain frequency and enlarged pain areas.