•Bus passenger balance measured for the first time in the real environment.•The design of the lower deck of the bus does not affect passenger balance.•On the bus, higher acceleration levels force all but older and female passengers to reduce double support time.•Passengers’ ability to control balance reduces with the increase of acceleration.•Middle-aged of both genders and men present more difficulties in controlling their balance in all cases. Travelling by bus is a way millions of people choose for their everyday activities. However, the large acceleration levels, and therefore the associated increased number of falls and non-collision injuries, force current users to shift to other modes of transport, with cars still remaining the preferred choice. This study investigates whether there is a possibility to improve the safety and comfort of buses, where all passengers can walk naturally inside a moving bus. Twenty-nine regular bus users, between 20 and 80 years old, were invited to participate in a series of experiments. Their natural gait whilst walking on a flat surface was monitored in a static laboratory and was compared to their gait whilst walking on the lower deck of a moving bus. The examined acceleration levels (low – 1.0 m/s2, medium – 1.5 m/s2, high – 2.5 m/s2) were set in the range of accelerations experienced by passengers on the real bus service in London. An ANOVA test was conducted on measures of changes in gait (double support time) as a measure of balance, taking into account passengers’ age and gender as well as the acceleration of the bus. The results revealed that, although the dimensions of the lower deck of the bus are narrow, passengers are still able to move to the back of the stationary bus whilst sustaining their natural balance. However, their ability to control balance reduces with the increase of acceleration.