The aim of this study was to determine the relative and absolute reliability of second lactate turnpoint using fixed and individual blood lactate method in competitive cyclists. Twenty-eight male, well-trained cyclists (30.2 ± 10.1 years, 72.0 ± 7.4 kg, 177.3 ± 4.7 cm) were recruited to participate in this study. Cyclists completed two incremental cycling tests to exhaustion over a period of 7 days to determine their peak power output, maximal oxygen uptake, maximal heart rate, maximal blood lactate concentration and two lactate turnpoint criteria. The fixed blood concentration criterion (3.5 mM) and an individual criterion were assessed by a lactate-power curve, considering power output, heart rate and oxygen uptake. The main finding of this study was that both lactate turnpoint criteria showed identical low within-subject variation for power output (2.8% coefficient of variation). High values for test-retest correlations ranging from r = 0.70 to r = 0.94 were found for all variables in both threshold criteria. In conclusion, the individual and fixed method to determine the second lactate turnpoint showed similar high absolute and relative reliability in competitive cyclists.