Appropriate cycling cleat adjustment could improve triathlon performance in both cycling and running. Prior recommendations regarding cleat adjustment have comprised aligning the first metatarsal head above the pedal spindle or somewhat forward. However, contemporary research has questioned this approach in triathlons due to the need to run immediately after cycling. Subsequently, moving the pedal cleat posteriorly could be more appropriate. This study evaluated the effectiveness of a triaxial accelerometer to determine acceleration magnitudes of the trunk in outdoor cycling in two different bicycle cleat positions and the consequential impact on trunk acceleration during running. Seven recreational triathletes performed a 20 km cycle and a 5 km run using their own triathlon bicycle complete with aerodynamic bars and gearing. Interpretation of data was evaluated based on cadence changes whilst triathletes cycled in an aerodynamic position in two cleat positions immediately followed by a self-paced overground run. The evaluation of accelerometer-derived data within a characteristic overground setting suggests a significant increase in total trunk acceleration magnitude during cycling with a posterior cleat with significant increases to longitudinal acceleration (p = 0.04) despite a small effect (d = 0.2) to the ratings of perceived exertion (RPE). Cycling with a posterior cleat significantly reduced longitudinal trunk acceleration in running and overall acceleration magnitudes (p < 0.0001) with a large effect size (d = 0.9) and a significant reduction in RPE (p = 0.02). In addition, running after cycling in a posterior cleat was faster compared to running after cycling in a standard cleat location. Practically, the magnitude of trunk acceleration during cycling in a posterior cleat position as well as running after posterior cleat cycling differed from that when cycling in the fore-aft position followed by running. Therefore, the notion that running varies after cycling is not merely an individual athlete’s perception, but a valid observation that can be modified when cleat position is altered. Training specifically with a posterior cleat in cycling might improve running performance when trunk accelerations are analysed.