Delayed onset muscle soreness (DOMS) is reduced when the same exercise is repeated after a certain interval. However, the mechanism for this adaptation, called a repeated bout effect, is still not well understood. Recently, we showed that upregulated nerve growth factor (NGF) triggered by B2 bradykinin receptor (B2R) activation in exercised muscle was responsible for DOMS. In this study, we investigated whether NGF upregulation was reduced after repeated bouts of exercise in rats, and if so, whether this change occurred upstream of B2R. A bout of 500 lengthening contractions (LC) was applied on day 0 and again 5 days later. DOMS was evaluated by the mechanical withdrawal threshold of the exercised extensor digitorum longus (EDL) muscle. Mechanical hyperalgesia and NGF mRNA upregulation in EDL were observed after the first LC, but not after the second LC. We then injected HOE140, a B2R antagonist with effects lasting only several hours, once before the first LC. This blocked the development of mechanical hyperalgesia and NGF mRNA upregulation not only after the first LC but also after the second LC. This suggests that adaptation occurred upstream of B2R, as the influence of the first LC was limited to that area by HOE140.