Purpose: Traditional physiological testing and monitoring tools have restricted our ability to capture parameters that best relate to cycling performance under variable-intensity race demands. This study examined the validity of a 1-h variable cycling test (VCT) to discriminate between different-performance-level cyclists. Methods: Ten male national- and 13 club-level cyclists (body mass, 67 [9] and 79 [6] kg; peak power output, 359 [43] and 362 [21] W, respectively) completed a VO2max test and two 1-h VCT protocols on 3 separate occasions. The VCT consisted of 10 × 6-min segments containing prescribed (3.5 W·kg−1) and open-ended phases. The open-ended phases consisted of 4 × 30–40 s of “recovery,” 3 × 10 s at “hard” intensity, and 3 × 6-s “sprint” with a final 10-s “all-out” effort. Results: Power output for the 6- and 10-s phases was moderately higher for the national- compared with club-level cyclists (mean [SD] 10.4 [2.0] vs 8.6 [1.6] W·kg−1, effect size; ±90% confidence limits = −0.87; ±0.65 and mean [SD] 7.5 [0.7] vs 6.2 [1.0] W·kg−1, effect size; ±90% confidence limits = −1.24; ±0.66, respectively). Power output for the final 10-s “all-out” sprint was 15.4 (1.5) for the national- versus 13.2 (1.9) W·kg−1 for club-level cyclists. Conclusion: The 1-h VCT can successfully differentiate repeat high-intensity effort performance between higher-caliber cyclists and their lower-performing counterparts.