International Journal of Exercise Science 13(3): 964-978, 2020. This study was designed to compare systemic O2 utilization (VȮ2), and changes in tissue O2 extraction [deoxyhemoglobin ($Δ$HHb)] in the vastus lateralis (VL), gastrocnemius (GAST) and pre-frontal cortex (PFC) tissue; between aerobically short-term trained (STT) and long-term trained (LTT) older men (40-60 yr) who were matched for current training load. On separate occasions, 14 STT and 14 LTT participants completed ramp incremental (RI) and square-wave constant load (SWCL) tests on a cycle ergometer. In LTT compared to STT; (i) VȮ2 was higher during the RI (p > 0.001) and SWCL (p >0.001) tests, (ii) $Δ$HHb in the GAST was greater in SWCL (p = 0.011); and (iii) $Δ$HHb in the PFC was greater at 90% GET during SWCL (p = 0.011). The additional years of training in LTT compared to STT (LTT 17.50yr ± 6.94yr vs STT 1.68yr ± 0.31yr) were associated with higher VȮ2peak, and sub-GET VȮ2, and $Δ$HHb in the GAST and PFC at sub-GET exercise, despite there being no difference in current training volume.