Osgood–Schlatter disease (OSD), a condition that affects adolescents, causes inflammation, pain, and prominence at the tibial tuberosity. The causes of OSD are not well understood, but eccentric contractions in the quadriceps have been suggested as a possible factor. To investigate this, a study was conducted in which 24 rats were divided into two groups: the downhill treadmill running (DR) group and the control (CO) group. The DR group underwent a preliminary running program for 1 week, followed by a main running program for 3 weeks. The results showed that the deep region of the tibial tuberosity in the DR group was larger than that in the CO group, and inflammatory cytokines involved in gene expression were upregulated in the DR group. The anterior articular cartilage and deep region in the DR group were also immunoreactive to substance P. Additionally, high-activity chondrocytes of small size were observed in the non-calcified matrix. Thus, the DR group exhibited symptoms similar to OSD, including inflammation, pain, and prominence. These findings suggest that eccentric contractions in the quadriceps may play a role in the development of OSD. Further research is needed to better understand the pathophysiology of this condition and develop effective treatment options.