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The purpose of this study was to investigate how treatment with triptorelin, a gonadotropin releasing hormone agonist, to block puberty in adolescent male rats affects their epigenetic regulation via global DNA methylation changes in skeletal muscle and to determine if physical activity has an effect on DNA methylation status during treatment. Twenty-four male, Sprague-Dewey rats were randomly assigned to different groups: 12 treated with 1 μg of triptorelin daily (PB group) while the other 12 received an equivalent volume of saline as a placebo (control, CON) for four weeks. In order to determine the interaction with physical activity, six PB rats and six CON rats were housed in cages with running wheels so they could be physically active (WR), while the other 12 rats were housed in cages without wheels as the sedentary group (SED). There were, therefore, four total groups (CON + SED, CON + WR, PB + SED, PB + WR). Following the treatment period, rats were euthanized, and the right soleus muscle was collected to isolate its DNA and analyze for global DNA methylation level using a MethylFlash Methylated DNA 5-mC Quantification Kit from Epigentek (Farmingdale, New York, U.S.). No significant drug nor physical activity effects were found on DNA methylation status, weight, weight gain, nor soleus weight. There were also no significant interactions for all parameters. Potential explanations are differentially methylated CpG islands canceling each other out, the short length of the treatment period, or the type of physical activity performed. The results do not support the hypothesis that triptorelin or physical activity alter global DNA methylation.