Background: To determine the validity of the lactate threshold (LT) and maximal oxygen uptake ( O2max) determined during graded exercise test (GXT) of different durations and using different LT calculations. Trained male cyclists (n = 17) completed five GXTs of varying stage length (1, 3, 4, 7 and 10 min) to establish the LT, and a series of 30-min constant power bouts to estab- lish the maximal lactate steady state (MLSS). O2 was assessed during each GXT and a subsequent verification exhaustive bout (VEB), and 14 different LTs were calculated from four of the GXTs (3, 4, 7 and 10 min)—yielding a total 56 LTs. Agreement was assessed between the highest V_ O2 measured during each GXT ( VO2peak) as well as between each LT and MLSS. V O2peak and LT data were analysed using mean difference (MD) and intraclass correlation (ICC). Results: The O2peak value from GXT1 was 61.0 ± 5.3 mL.kg-1.min-1 and the peak power 420 ± 55 W (mean ± SD). The power at the MLSS was 264 ± 39 W. O2peak from GXT3, 4, 7, 10 underesti- mated VO2peak by ~1–5 mL kg-1.min-1 Many of the traditional LT methods were not valid and a newly developed Modified Dmax method derived from GXT4 provided the most valid esti-mate of the MLSS (MD = 1.1 W; ICC = 0.96). Conclusion: The data highlight how GXT protocol design and data analysis influence the determination of both O2peak and LT. It is also apparent that V_ O2max and LT cannot be determined in a sin-gle GXT, even with the inclusion of a VEB.