ObjectivesThe purpose of this study was to analyze the time measurement validity and reliability (between raters and test–retest) of the 4 × 10-m shuttle run test to assess motor fitness in adults, according to gender, age, and physical activity levels. DesignCross-sectional. A total of 230 adults (86 women) aged 18–64 years participated in the study. MethodsThe time taken to complete the 4 × 10-m shuttle run test was recorded simultaneously by a trained and an untrained rater (inter-rater reliability) and by photoelectric cells (time measurement validity). 48–72 h later, the test was repeated under the same conditions (test–retest reliability). ResultsThe systematic error for trained rater vs. photocell was close to zero (0.0125, p < 0.01), with an effect size of 0.006; and for both, untrained rater vs. photocell and trained rater vs. untrained rater was ∼0.2 s (p < 0.001) with an effect size of 0.09. For the test–retest reliability, the systematic error was 0.05 s (p < 0.001), with an effect size of 0.26, the intraclass correlation coefficient was 0.998 and the coefficient of variation reported a variability of 0.73 %. Results were not influenced by gender and age, while these improved for active vs. non-active participants. ConclusionsFindings indicate that measurements with trained raters are a valid and reliable method for assessing the 4 × 10-m shuttle run test in adults. It is highly recommended that raters be trained to minimize the measurement error.