This study aimed to analyze the effects of padel match play on circulating substrates, metabolites, energy balance enzymes, and muscle damage biomarkers and evaluate possible sex-related differences. Twenty-two trained padel players (13 female and 9 male young-adult players) were recruited for this study in which simulated padel matches were analyzed. Circulating levels of substrates (glucose -BG- and triglycerides -TGs-), metabolites (creatinine -Cr- and urea), energy balance enzymes (lipoprotein lipase -LPL-), and muscle damage biomarkers (creatine kinase -CK-, lactate dehydrogenase -LDH-, and fatty acid-binding protein 3 -FABP-3-) were assessed both pre- and post-padel competition. Time analysis of padel matches reported a real time–total time ratio of 0.4. Moreover, players’ mean heart rate during padel matches represented around 75% of their individual maximum value. Unaltered BG levels and a slight decrease in TGs were observed post-exercise. Cr, urea, LPL, CK, LDH, and FABP-3 levels increased after padel matches when total group was considered. Moreover, sex-related differences in Cr, CK, and LDH blood concentrations were found in both pre- and post-padel competition. According to our results, the padel competition could be defined as a low- or moderate-impact sport in which aerobic energy system contribution is prevalent although anaerobic metabolism also plays a key role in performing padel shots and other explosive actions. Considering that male and female players exercised at the same relative intensity during padel matches, sex differences found in muscle damage biomarkers could be due to the greater muscle mass in males.