The Assessing Levels of Physical Activity (ALPHA) health-related fitness test battery is a set of reliable, valid, and feasible tests to assess health-related physical fitness in children and in adolescents. The aim of this study was to examine the reliability of this battery in adolescents with Down syndrome (DS). The extended ALPHA health-related fitness test battery was performed twice within 1 month in 17 apparently healthy adolescents, aged 12-18 years, with DS who had an intelligence quotient ≥ 35. Intraclass correlation coefficient (ICC) was used to determinate test-retest reliability, and nonparametric Wilcoxon signed rank test was used to compare mean differences between measurements. With the exception of subscapular skinfold test, which obtained a moderate agreement (ICC = 0.64), all tests had a very high reliability: the 20-m shuttle-run test (ICC = 0.86), the right handgrip strength test (ICC = 0.86), the left handgrip strength test (ICC = 0.86), the standing broad jump test (ICC = 0.85), body mass index (ICC = 0.95), waist circumference (ICC = 0.98), triceps skinfold (ICC = 0.85), and the 4 × 10-m shuttle-run test (ICC = 0.92). There were no significant differences (all p > 0.05) in any of the tests. The ALPHA health-related fitness battery is reliable for measuring health-related components of fitness in adolescents with DS.