Purpose: To investigate biomechanical and physiological responses to soccer-specific exercise incorporating an extra time period (ET) and assess the test-retest reliability of these responses. Methods: Twelve soccer players performed 120 min of soccer-specific exercise. Tri–axial (PLTotal) and uni-axial PlayerLoad™ in the vertical (PLV), anterior-posterior (PLA-P) and medial-lateral (PLM-L) planes were monitored using a portable accelerometer. Likewise, respiratory exchange ratio (RER) was recorded throughout exercise. At the end of each 15 min period, players provided differential ratings of perceived exertion ([d-RPE]) for legs [RPE-L], breathlessness [RPE-B] and overall [RPE-O]) and capillary samples were taken to measure blood lactate (BLa) concentrations. The soccer-specific exercise was completed twice within seven days to assess reliability. Results: A main effect for time was identified for PLTotal (p = 0.045), PLV (p = 0.002), PLA-P (p = 0.011), RER (p = 0.001), RPE-L (p = 0.001), RPE-O (p = 0.003) and CMJ (p = 0.020). A significant increase in PLTotal (234 ± 34 a.u) and decrease in RER (0.87 ± 0.03) was evident during 105-120 min versus 0-15 min (215 ± 25 a.u; p = 0.002 and 0.92 ± 0.02; p = 0.001). Coefficient of variations were <10% and Pearson’s correlation coefficient demonstrated moderate to very strong (0.33-0.99) reliability for all PL variables, RPE-B, BLa and RER. Conclusions: These results suggest that mechanical efficiency is compromised and an increased rate of lipolysis is observed as a function of exercise duration, particularly during ET. This data has implications for practitioners interested in fatigue-induced changes during ET.