Peripheral blood mononuclear cells (PBMCs) represent a heterogeneous mix of cells with paracrine functions that may be altered following prolonged exercise. We determined the effect of ultramarathon running on PBMC paracrine function and PBMC subtype number. Recreational athletes participated in a 50 km ultramarathon. Blood was sampled from N = 7 at baseline, 10 km, 50 km, and 24 h post-race. PBMCs were isolated and cultured, and conditioned media was used for a HUVEC-based proliferation assay. CD31+, CD3+, and CD31+/CD3+ PBMCs were quantified at each time point. Proliferation increased from baseline to 50 km (p = 0.004) and was reduced from 50 km to 24 h post (p = 0.008). There was an increase in CD31+ PBMCs after 50 km (p = 0.014), returning to baseline at 24 h post-race (p = 0.246). CD3+ PBMC and CD31+/CD3+ PBMC numbers were reduced after 50 km (p = 0.001 and p = 0.002, respectively), returning to baseline levels 24 h post-race (p = 0.190 and p = 0.315, respectively). PBMC paracrine activity following a 50 km enhances endothelial cell proliferation. Alterations in PBMC subtypes after 50 km suggest a protective role of PBMCs in response to prolonged stresses of ultramarathon running.