With ride quality being one of the most sought-after characteristics of a road bicycle by customers as well as by bicycle manufacturers, the vibrational behaviour of the bicycle/cyclist system has grown into an active field in sport engineering research in recent years. When assessing bicycle transmissibility and ride comfort, it is important to control test conditions to obtain repeatable load and acceleration measurements at the cyclist’s contact points with the bicycle. Surprisingly, however, this consideration has not yet been specifically addressed in the literature. The aim of this paper is a first effort to investigate the effect of a selected set of test conditions on the measurement of vibration induced to the cyclist by a road bicycle. Our results showed that all the test conditions selected had a significant effect on the level of vibration induced to the cyclist.