Background To determine the validity of the lactate threshold (LT) and maximal oxygen uptake (V _ O 2max) 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 establish the maximal lactate steady state (MLSS). V _ O 2 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 _ O 2 measured during each GXT (V _ O 2peak) as well as between each LT and MLSS. V _ O 2peak and LT data were analysed using mean difference (MD) and intraclass correlation (ICC). Results The V _ O 2peak value from GXT 1 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. V _ O 2peak from GXT 3, 4, 7, 10 underestimated V _ O 2peak by ~1–5 mL .kg -1.min -1. Many of the traditional LT methods were not valid and a newly developed Modified D max method derived from GXT 4 provided the most valid estimate 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 V _ O 2peak and LT. It is also apparent that V _ O 2max and LT cannot be determined in a single GXT, even with the inclusion of a VEB.