We investigated the effects of a two-week run sprint interval training (SIT) intervention on 3000m performance, mitochondrial and microvascular function, maximal oxygen uptake (VO2max) , oxygen uptake (VO2p) kinetics, and running economy (RE) in trained runners. Further, at baseline mitochondrial and microvascular function were compared between the medial gastrocnemius (GM), tibialis anterior (TA), and vastus lateralis (VL) muscles. Twenty-four runners were recruited and assigned to either SIT (six sessions of four to six 30s sprints) or CON (maintain regular endurance training). The study found that at baseline, subjects had better mitochondrial function in the GM compared to the VL, while microvascular function was better in the GM and TA than in the VL. Post intervention, a non-significant improvement (2.8%, P = 0.10) in 3000m performance was observed in the SIT group, while no other changes were observed in either group. In trained runners, SIT may improve aerobic performance, independently of changes in VO2max, VO2p kinetics, RE, mitochondrial and microvascular function.