Cycling can bring a wide range of social, economic, and health benefits to individuals and communities. The safety and efficiency of bicycle facilities can be significantly impacted by the interactions among riders. This study aims to examine the microscopic characteristics of how cyclists interact with each other when they are in a single file movement based on the trajectory data collected from an experiment. Reaction delay was obtained by optimizing the correlation between relative speed and acceleration curves for individual cyclists and it was found that even for a given cyclist, this characteristic time delay could vary considerably, and be situation-dependent. Furthermore, it was found that the distribution of reaction delay, which has an average (±SD) of 0.66 s (±0.33 s), followed a log-normal distribution. The strong correlation observed between relative speed and time-delayed acceleration resembles the behavior observed in car-following situations, highlighting that relative speed is an essential factor influencing the acceleration behavior of cyclists. Multiple linear regression models were used to understand the association between acceleration and other key microscopic variables, e.g., spacing and relative speed, which are commonly used in microscopic behavior models. While the spacing between cyclists was found to have a significant impact on acceleration behavior, its effect was not as significant as that of relative speed. The outcomes of this study provide valuable insights into the cyclists’ behavior and can aid in the development of microscopic simulation models.