Running has become increasingly popular worldwide. Among runners, there exists a wide range of expertise levels. Investigating the differences between runners at two extreme levels, that is novices and experts, is crucial to understand the changes that occur as a result of multiple years of training. Vertical oscillation of center of mass (CoM), stride frequency normalized to the leg length, and duty factor, which describes the step time relative to the flight time, are key biomechanical parameters that have been shown to be closely related to the running economy and are used to characterize the running style. The variability characteristics of these parameters may reveal valuable information concerning the control of human locomotion. However, how the expertise level and running speed affect the variability of these key biomechanical parameters has not yet been investigated. The aim of this study was to analyze the effects of expertise level (novice vs. expert) and running speed (10 km/h vs. 15 km/h) on these parameters and their variability. It was hypothesized that expert runners would have lower vertical oscillation of CoM, normalized stride frequency, and duty factor and show less variability in these parameters. The parameters’ variability was operationalized by the coefficient of variation. The mean values and variability of these key biomechanical parameters according to expertise level and running speed were compared with rmANOVAs. The results showed that the experts had a lower duty factor and less variable vertical oscillation of CoM and normalized stride frequency, independently of the running speed. At a higher running speed, the variability of vertical oscillation of CoM was higher, whereas that of normalized stride frequency and duty factor did not change significantly. To the best of our knowledge, this is the first study analyzing the effects of expertise level and running speed on the variability of key biomechanical parameters.