This study aimed to examine the effect of running velocity on spatiotemporal parameters and lower-body stiffness of endurance runners, and the influence of the performance level on those adaptations. Twenty-two male runners (novice [NR], n = 12, and elite runners [ER], n = 10) performed an incremental running test with a total of 5 different running velocities (10, 12, 14, 16, 18 km/h). Each condition lasted 1 min (30 s acclimatization period, and 30 s recording period). Spatiotemporal parameters were measured using the OptoGait system. Vertical (Kvert) and leg (Kleg) stiffness were calculated according to the sine-wave method. A repeated measures ANOVA (2 x 5, group x velocities) revealed significant adaptations (p < 0.05) to increased velocity in all spatiotemporal parameters and Kvert in both NR and ER. ER showed a greater flight time (FT) and step angle (at 18 km/h) (p < 0.05), longer step length (SL) and lower step frequency (SF) (p < 0.05), whereas no between-group differences were found in contact time (CT) nor in the sub-phases during CT at any speed (p ≥ 0.05). ER also showed lower Kvert values at every running velocity (p < 0.05), and no differences in Kleg (p ≥ 0.05). In conclusion, lower SF and Kvert and, thereby, longer FT and SL, seem to be the main spatiotemporal characteristics of high-level runners compared to their low-level counterparts.