Purpose <p>To investigate the effects of cyclic stretch on the differentiation of rat adipose-derived stem cells (rADSCs) into chondrocytes, focusing on the effect of cyclic stretch at two different frequencies on chondrogenesis.</p> Method <p>The rADSCs chondrogenesis was induced by the growth factor transforming growth factor-β1 and cyclic uniaxial stretching at 0.5&#xa0;Hz and 1&#xa0;Hz. The degree of chondrogenic differentiation was evaluated in terms of various indicators. The impacts of two cyclic stretch frequencies were compared in terms of cell morphology, cartilage staining, glycosaminoglycan (GAG) content, protein expression, and the expression of specific genes.</p> Results <p>The effects of stimulation at different frequencies on cell chondrogenesis varied. Cyclic stretch at 0.5&#xa0;Hz showed a more significantly enhanced rADSCs chondrogenesis, as evidenced by morphology, staining, GAG contents, and protein expression. The effect of cyclic stretch frequency on rADSCs chondrogenesis was further evaluated in the context of cell apoptosis. While cyclic stretch at 1.0&#xa0;Hz led to cell apoptosis, that at 0.5&#xa0;Hz reduced apoptosis, promoting chondrogenesis.</p> Conclusion <p>This study offers insight into the effects of cyclic stretch on rADSCs chondrogenesis and offers a key approach for treating cartilage diseases, such as osteoarthritis.</p> Trial registration <p>NA.</p>