The purpose of this study was to calculate and validate reference standards for the 20-m shuttle run test (SR) in youths aged 10-18 years. Reference standards based on the number of completed SR laps were calculated by LMS method in a reference group of 5559 students. Cut-off values for SR laps were determined and tested by ROC curve analysis in a validation group (633 students), from which waist circumference, HDL-cholesterol, triglycerides, fasting glucose and mean arterial pressure were assessed to calculate a metabolic risk score, later dichotomized in low and high metabolic risk (HMRS). The accuracy of SR laps standards was significant for girls (AUC = 0.66; 95% CI = 0.58-0.74; p < .001) and boys (AUC = 0.71; 95% CI = 0.62-0.79; p < .001) for identifying subjects at HMRS. The 40th percentile was the best cut-off for SR laps in girls (SENS = 0.569; 1-SPEC = 0.330) and boys (SENS = 0.634; 1-SPEC = 0.266). New SR laps reference standards are able to discriminate metabolic risk levels, and may provide a valuable tool for early prevention of cardiovascular risk factors.