We examined the influence of competitive standard of the opponent on activity profile and fatigue during preseason friendly soccer matches. Time motion analysis was performed in a male professional soccer team (N = 14) during six friendly games played against professional, semi-professional and amateur-level opponents (PL, SPL and AL). The reference team covered higher acceleration distance, acceleration and deceleration > 2 m· s−2 distance against PL than AL (ES = 0.77 to 0.91). Acceleration and deceleration distance > 2 m· s−2 was also higher (ES = 0.66 to 0.84) against SPL than AL. Greater decreases in total distance, distance> 16 km· h−1 and > 22 km· h−1, total acceleration and deceleration, acceleration and deceleration distance > 2 m· s−2 (ES = 0.84 to 2.20) were also observed during PL compared to AL opponent. Playing against a stronger opponent seems to be more physically demanding, with special emphasis on events related with change of velocity (accelerations and decelerations). Declines in physical performance appear more evident against a higher opponent.