The purpose of this study was to examine the effect of increment durations of 1-min and 4-min during progressive incremental exercise tests on: 1) the distribution of lactate between plasma and red blood cells (RBCs), and 2) lactate threshold (LT) detection via three conventional methods using whole blood lactate concentration ([La]) or plasma [La]. Eight males (age, 22.5± 0.6 yr; height, 170.6 ± 2.3 cm, weight, 76.0 ± 3.1 kg, and ˙VO2peak, 42.8 ± 2.0 mL·kg-1·min-1) performed two progressive load tests to volitional fatigue on a cycle ergometer. Work rate was increased 30 W at 1-min or 4-min intervals. All data were normalized to individual LT work rates. For both protocols, whole blood [La], plasma [La], RBC [La], and [La] gradient increased significantly (P < 0.05) after exercise intensity exceeded LT. However, the RBC:plasma [La] ratio remained at the resting value throughout the progressive exercise tests. The increase in [La] gradient after LT, with no change in the RBC:plasma lactate ratio, suggests that given an incremental work rate increase of 30 W, 1 min is adequate for equilibration of lactate between the plasma and RBCs. Also, under the conditions of this investigation, neither blood fraction analyzed nor exercise protocol had any effect on estimations of LT (in terms of ˙VO2) by the Visual and Log-Log methods. However, LT determined by a fixed [La] of 2 mM may underestimate LT when plasma samples are used.