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Players in team sports like soccer often make acceleration and deceleration movements, which are more energetically demanding than running at constant speed. The first aim of the present study was to estimate this difference in associated energy cost. To this end, we compared the actual energy cost of shuttle running to that of running at constant speed. In addition, since measuring oxygen consumption is not feasible during soccer, the study’s second aim was to determine the validity of an indirect approach to estimate energy cost provided by di Prampero et al. (2005) using time–motion data obtained from a tracking system as input.Fourteen male amateur soccer players performed aerobic constant and continuous shuttle running at six different speeds (range = 7.5–10.0 km·h−1) on artificial turf. Measured energy cost was compared to the energy cost estimated with di Prampero’s (2005) equation using data from a local position measurement (LPM) system as input.As expected, measured energy cost was significantly higher (∼30%–50%) for shuttle running than for constant running (P < 0.001), and this difference increased with speed. For constant running, estimated energy cost was significantly higher (6%–11%) than measured energy cost, whereas for shuttle running, estimated energy cost was significantly lower (−13% to −16%) than measured energy cost.Shuttle running raised the player’s energy cost of running compared to constant running at the same average speed. Although actual energy cost of constant running was significantly overestimated by di Prampero’s approach using LPM data as input, actual energy cost of shuttle running was significantly underestimated.