The purpose of the investigation was to observe how varying occlusion durations affected neuromuscular activation and microvascular oxygenation during low-volume isometric knee extension exercise. Healthy, recreationally active males performed isometric knee extension at a variety of submaximal intensities under different blood flow restriction (BFR) occlusion durations. The occlusion pressure (130% SBP) was applied either 5 min prior to exercise (PO), immediately prior to exercise (IO) or not during exercise (CON). Surface electromyography (sEMG) and near-infrared spectroscopy (NIRS) was used to record the neuromuscular activation and microvascular oxygenation of the knee extensors during exercise. No difference in sEMG was observed in the vastus lateralis or vastus medialis during any exercise condition or any submaximal intensity. PO elicited greater microvascular deoxygenation (deoxy-[Hb + Mb]) compared to CON (P≤0·05) at all submaximal intensities and also compared to IO at 20% maximal voluntary contraction (MVC). IO resulted in a greater deoxy-[Hb + Mb] response during low-intensity exercise (20% and 40% MVC) compared to CON (P≤0·05). These findings suggest that applying BFR 5 min before exercise can enhance the exercise-induced metabolic stress (i.e. deoxy-[Hb + Mb]), measured via NIRS, during low-intensity exercise (20% MVC) compared to applying BFR immediately prior to exercise. Furthermore, the increased metabolic stress observed during IO is attenuated during high-intensity (60% MVC, 80% MVC) exercise when compared to CON conditions. Knowledge of the changes in exercise-induced metabolic stress between the various occlusion durations may assist in developing efficient BFR exercise programmes.