We examined physiological responses and fatigue in adolescent boys and girls during a repeated shuttle-sprint running test in a school setting. We also compared three calculation methods to assess fatigue during repeated sprints: the fatigue index (FI), the percent sprint decrement (Sdec), and the slope of performance decrement (SlopeD). Twenty-six adolescent students (10 girls and 16 boys, age: 15.3 ± 0.5 y) performed six 30 m sprints with a 180° change of direction at 15 m, interspersed with 10 s of recovery. Blood lactate (BL), heart rate (HR) and countermovement jump performance (CMJ) were measured before and after the sprint test. Boys achieved significantly faster sprint times (11.7% to 14.8% faster than girls in all sprints,p< 0.01), and had higher post-test BL compared with girls (14.0 ± 2.9 vs. 11.3 ± 3.2 mmol/L;p= 0.02; d = 0.93). However, all fatigue indices (FI, Sdec and SlopeD) were similar in boys and girls (FI = 11.5±5.0 vs. 11.5±5.0; SlopeD = 10.6±4.8 vs. 14.5±5.1; Sdec = 5.6±2.2 vs. 7.3±2.2, in boys and girls, respectively,p> 0.05 for all) and were highly correlated with each other (r = 0.86 to 0.97,p< 0.01). CMJ was higher in boys (p< 0.05), but CMJ performance dropped similarly from pre- to post-test in boys and girls (13.7 ± 7.0%,p< 0.01). In conclusion, neuromuscular fatigue during and after repeated shuttle-sprint running is similar in boys and girls, despite the faster sprint times, higher CMJ and higher BL in boys. SlopeD may be used as an alternative index to quantify fatigue during repeated shuttle-sprinting, as it takes into account performance in all sprints and may provide an average sprint-by-sprint performance drop.