Objective: The number of e-bike users has increased significantly over the past few years and with it the associated safety concerns. Because e-bikes are faster than conventional bicycles and more prone to be in conflict with road users, e-bikers may need to perform avoidance manoeuvres more frequently. Braking is the most common avoidance manoeuvre, but is also a complex and critical task in emergency situations, since cyclists must reduce speed quickly without losing balance. The aim of this study is to understand the braking strategies of e-bikers in real-world traffic environments and to assess their road safety implications. This paper investigates 1) how cyclists on e-bikes use front and rear brakes during routine cycling, and 2) whether this behaviour changes during unexpected conflicts with other road users. Methods: Naturalistic data were collected from six regular bicycle riders who each rode e-bikes during a period of two weeks, for a total of 32.5 hours of data. Braking events were identified and characterized through a combined analysis of brake pressure at each wheel, velocity, and longitudinal acceleration. Furthermore, the braking patterns obtained during unexpected events were compared with braking patterns during routine cycling. Results: In the majority of braking events during routine cycling, cyclists used only one brake at a time, favouring one of the two brakes according to a personal pre-established pattern. However, the favoured brake varied among cyclists: 66% favoured the rear brake and 16% the front brake. Only 16% of the cyclists showed no clear preference, variously using rear brake, front brake, or combined braking (both brakes at the same time), suggesting that the selection of which brake to use depended on the characteristics of the specific scenario experienced by the cyclist rather than on a personal preference. In unexpected conflicts, generally requiring a larger deceleration, combined braking became more prevalent for most of the cyclists; still, when combined braking was not applied, cyclists continued to use the favoured brake of routine cycling. Kinematic analysis revealed that, when larger decelerations were required, cyclists more frequently used combined braking instead of single braking. Conclusions: The results provide new insights into the behaviour of cyclists on e-bikes and may provide support in the development of safety measures including guidelines and best practices for the optimal brake use. The results may also inform the design of braking systems intended to reduce the complexity of the braking operation.