The fatigue provisions for bridge design in North America and Europe are highly simplified for the sake of practical implementation, in comparison with the state of the art in understanding of the true fatigue behavior. Historically, one significant simplification in the calibration of damage equivalence factors has been to assume that trucks cross the bridge one at a time, when in reality trucks are known to platoon, and multiple truck presence is not uncommon on longer span structures with higher traffic volumes. While code writers have taken steps more recently to consider multiple presence effects, these efforts have been limited, and a clear understanding of the influence of key design parameters on multiple presence effects has not yet been fully established. Against this background, the present work considers real-time multilane weigh-in-motion (WIM) data and compares fatigue damage from recorded loading events over years to the damage resulting from the same vehicles crossing the bridge individually, to better understand the significance of multiple vehicle presence effects on fatigue damage. For both finite and infinite life fatigue design, the results show that multiple vehicle presence effects can be significant, depending on traffic volume, road configuration, and influence line characteristics. Multiple presence factors are therefore defined, allowing practitioners to consider multiple presence effects appropriately when designing bridges where they may be relevant.