Recent studies have suggested that the capability to resist deterioration of physiological characteristics could be an independent factor contributing to endurance performance. This study aimed at investigating whether prolonged low-intensity exercise induces shifts in the lactate threshold, and whether fatigue-induced changes differ between the sexes. A total of 31 (15 females) recreational runners performed an incremental treadmill test and a 90-min low-intensity exercise (LIT90) on two separate occasions. The LIT90 was performed at 90% of the first lactate threshold speed (LT1v), derived from the incremental treadmill test. The LT1v was determined from a 5-stage (3min) submaximal threshold test (SubmaxLT), performed before and after LIT90. The SubmaxLTs were followed by a 10/5 reactivity jump test. Respiratory gases, heart rate (HR), and HR-derived detrended fluctuation analysis alpha 1 (DFA-a1) were assessed every 15min during the LIT90. A significant decrease (p < 0.01) was observed in the LT1v in females (-5.8 ± 4.4%) and in males (-5.3 ± 6.4%). The HR increased (p < 0.001) similarly in females (5.9 ± 3.1%) and in males (5.5 ± 3.6%) during the LIT90, while energy expenditure increased (3.1 ± 4.5%, p = 0.013) in females but remained unchanged in males (0.9 ± 3.1%). Change in DFA-a1 during the LIT90 was the only marker that correlated significantly with the relative change of LT1v (r = 0.463, p = 0.013). LIT90 induced significant decreases in the LT1v, and the changes were comparable between sexes. DFA-a1 could be a potential intra-session marker of durability.