Objective: To examine the reliability of a set of health-related physical fitness tests used in the European Union-funded Healthy Lifestyle in Europe by Nutrition in Adolescence (HELENA) Study on lifestyle and nutrition among adolescents. Design: A set of physical fitness tests was performed twice in a study sample, 2 weeks apart, by the same researchers. Participants: A total of 123 adolescents (69 males and 54 females, aged 13.6 +/- 0.8 years) from 10 European cities participated in the study. Measurements: Flexibility, muscular fitness, speed/agility and aerobic capacity were tested using the back-saver sit and reach, handgrip, standing broad jump, Bosco jumps (squat jump, counter movement jump and Abalakov jump), bent arm hang, 4 x 10 m shuttle run, and 20-m shuttle run tests. Results: The ANOVA analysis showed that neither systematic bias nor sex differences were found for any of the studied tests, except for the back-saver sit and reach test, in which a borderline significant sex difference was observed (P 0.044). The Bland-Altman plots graphically showed the reliability patterns, in terms of systematic errors (bias) and random error (95% limits of agreement), of the physical fitness tests studied. The observed systematic error for all the fitness assessment tests was nearly 0. Conclusions: Neither a learning nor a fatigue effect was found for any of the physical fitness tests when repeated. The results also suggest that reliability did not differ between male and female adolescents. Collectively, it can be stated that the reliability of the set of physical fitness tests examined in this study is acceptable. The data provided contribute to a better understanding of physical fitness assessment in young people.