Purpose: The aim of this study was to compare peak acceleration, deceleration and speed between preferred and non-preferred legs during the 5-0-5 change-of-direction test in adult male soccer players. Methods: The cross-sectional study design included twenty elite male soccer players (21.6 ± 2.0 years; 73.2 ± 6.1 kilograms; 174.8 ± 4.5 centimeters). The players were assessed in the 5-0-5 change-of-direction test during a single day between 3 p.m and 6 p.m. The assessments occurred on synthetic turf and were preceded by a 48-hour period of rest. Before the assessment players reported an average of 8 (“well recovered/somewhat energetic”) on the Total Quality Recovery scale. After indicating their preferred and non-preferred legs, players performed three trials on each leg, interspaced by 5 minutes between sets and 3 minutes between repetitions. Peak acceleration, peak deceleration and peek speed was recorded for each trial using a Polar Team Pro (Polar Electro, Kempele, Finland) device sampling at 10 Hz housed in a specialized pocked on the players upper backs. The best peak acceleration, deceleration, and speed for preferred and non-preferred legs was used for analysis. Coefficient of variations (CVs) were calculated for reliability, with paired sample t-tests and Cohen’s D effect sizes calculated to compare for differences between conditions. Results: CVs for peak acceleration were 3.6 ± 2.4% for preferred and 3.2 ± 1.7% for non-preferred leg, –4.2 ± 3.7% and –4.3 ± 3.2% for peak deceleration, 2.3 ± 1.3 and 2.0 ± 1.1% for peak speed. Comparisons revealed a significantly greater peak acceleration (+0.22 m/s2; t=5.131; p<0.001; d=0.581) and deceleration (+0.17 m/s2; t=–3.266; p=0.004; d=–0.586) for the non-preferred leg. While there were no significant differences found between legs for peak speed (t=1.142; p=0.268; d=0.298). Conclusions: Braking with the preferred or non-preferred leg during the 5-0-5 change-of-direction test appears to have an effect on peak acceleration and deceleration. It is therefore recommended, that when utilizing this test, standardization is needed in the leg chosen for braking and comparisons only made between preferred or non-preferred legs.