Blunted post-occlusive reactive hyperemia (PORH) after prolonged sitting (PS) has been used as evidence of microvascular dysfunction. However, it has not been determined if confounding variables are responsible for the reduction in PORH after PS. Therefore, the purpose of this study was to examine the PS-mediated changes in cardiovascular and metabolic factors that affect PORH using artificial intelligence (AI). We hypothesized that calf muscle metabolic rate (MMR) is attenuated after PS, which may reduce tissue hypoxia during an arterial occlusion (i.e., oxygen deficit) and PORH. Thirty-one subjects (male = 13, female = 18) sat for 2.5 h. A rapid-inflation cuff was placed around the thigh above the knee to generate an arterial occlusion. PORH was represented by the reoxygenation rate (RR) of the near-infrared spectroscopy (NIRS) tissue oxygenation index (TOI) after 5-min of arterial occlusion. An artificial intelligence model (AI) defined the stimulus-response relationship between the oxygen deficit (i.e., ΔTOI and TOI deficit), and RR with 65 previous PORH recordings. If the AI predicts the experimental RRs, then the change in RR is related to the change in the oxygen deficit. RR (Δ -0.27 ± 0.55 lnTOI%·s, = 0.001), MMR (Δ -0.46 ± 0.61 lnTOI%·s, < 0.001), ΔTOI (Δ -0.34 ± 0.62 lnTOI%, < 0.001), and the TOI deficit (Δ -0.42 ± 0.68 lnTOI%·s, < 0.001) were reduced after PS. In addition, strong linear associations were found between MMR and the TOI deficit ( = 0.900, < 0.001) and ΔTOI ( = 0.871, < 0.001). Furthermore, the AI accurately predicted the RRs pre- and post-PS ( = 0.471, = 0.328, respectively). Therefore, blunted PORH after PS may be caused by attenuated MMR and not microvascular dysfunction. Prolonged sitting reduces lower leg skeletal muscle metabolic rate in healthy individuals. Artificial intelligence revealed that impaired post-occlusive reactive hyperemia after prolonged sitting is related to a reduced stimulus for vasodilation and may not be evidence of microvascular dysfunction. Current post-occlusive reactive hyperemia protocols may be insufficient to assess micro- and macrovascular function after prolonged sitting.