The aim of this study was to evaluate the effect of acceleration on the external and internal load during repeated sprint exercises (RSE). This study used a cross-over design. Sixteen soccer players were included (mean ± SDs: age 21 ± 1 years; weight 71.1 ± 7.7 kg). RSE was 3 sets of 7 × 30 m sprints with 25 s and 3 min recovery between sprints and sets, respectively. RSE was performed using two protocols requiring either 10 m maximal acceleration (2.12 m.s−2 [RSE-MA]) or 10 m submaximal acceleration (1.66 m.s−2 [RSE-SA]). Global positioning systems (10 Hz; STATSports, Viper) were utilized to collect: high speed running (HSR), dynamic stress load (DSL), Heart Rate (HR) peak, time >85% HR peak, respiratory (RPEres) and muscular (RPEmus) rating of perceived exertion. RSE-MA induced higher load than RSE-SA in HSR (p = 0.037, ES = 0.20), DSL (p = 0.027, ES = 0.43), HR peak (p = 0.025, ES = 0.47), Time >85% HR peak (p = 0.028, ES = 1.11), RPEres (p = 0.001, ES = 1.10), and RPEmus (p = 0.001, ES = 0.73). This study shows that a different acceleration intensity in a RSE (MA vs. SA) impacts external and internal training load parameters.