Purpose: Aerobic endurance training (ET) increases systemic and peripheral oxygen utilisation over time, the adaptation pattern not being linear. However, the timing and mechanisms of changes in oxygen utilisation, associated with training beyond one year are not known. This study tested the hypothesis that in women aged 40–60 years performing the same current training load; systemic O2 utilisation (VO2) and tissue deoxyhaemoglobin (HHb) in the Vastus Lateralis (VL) and Gastrocnemius (GAST) would be higher in long term trained (LTT; > 5 yr) compared to a short term trained (STT; 6–24 months) participants during ramp incremental (RI) cycling, but similar during square-wave constant load (SWCL) cycling performed at the same relative intensity (below ventilatory turn point [VTP]); and that pre-frontal cortex (PFC) HHb would be similar between participant groups in both exercise conditions. Methods: Thirteen STT and 13 LTT participants performed RI and SWCL conditions on separate days. VO2, and VL, GAST, and PFC HHb were measured simultaneously. Results: VO2peak was higher in LTT compared to STT, and VO2 was higher in LTT at each relative intensities of 25%, 80% and 90% of VTP in SWCL. HHb in the VL was significantly higher in LTT compared to STT at peak exercise (4.54 ± 3.82 vs 1.55 ± 2.33 μM), and at 25% (0.99 ± 1.43 vs 0.04 ± 0.96 μM), 80% (3.19 ± 2.93 vs 1.14 ± 1.82 μM) and 90% (4.62 ± 3.12 vs 2.07 ± 2.49 μM) of VTP in SWCL. Conclusions: The additional (12.9 ± 9.3) years of ET in LTT, resulted in higher VO2, and HHb in the VL at peak exercise, and sub—VTP exercise. These results indicate that in women 40–60 years old, systemic and muscle O2 utilisation continues to improve with ET beyond two years.