Acute exercise alters the surface expression of toll-like receptors (TLRs) and HLA.DR on blood monocytes, which could transiently compromise immunity. As serum factors might be responsible, we examined the effects of autologous post-exercise serum exposure on TLR2, TLR4 and HLA.DR expression on resting blood monocytes and their subtypes. Eight trained cyclists completed an ergometer 60 km time trial. PBMCs and serum were obtained before, immediately after and 1 h after exercise. TLR2, TLR4 or HLA.DR expression (gMFI) was determined on blood monocyte subtypes expressing combinations of CD14 and CD16 by flow cytometry, and on resting monocytes exposed to 50% autologous serum (pre, immediately after or 1 h after exercise) for 18 h in culture. Immediately after exercise, total monocyte expression of TLR2 and TLR4 increased by 41 and 27%, respectively, while HLA.DR expression was 39% lower than baseline. TLR2 and TLR4 was 53 and 84% greater 1 h after exercise, respectively, while HLA.DR was 48% lower. Changes in TLR2 and TLR4 expression occurred on the CD14++bright/CD16+dim monocyte subtype only, while HLA.DR expression changed on the CD14+dim/CD16++bright subtype. Serum did not affect monocyte TLR2 or TLR4 expression but 1 h post serum increased expression of HLA.DR on total monocytes and the CD14+dim/CD16++bright subtype, which was in contrast to the change observed at this time after exercise. We conclude that a bout of strenuous aerobic exercise alters the surface expression of TLR2, TLR4 and HLA.DR on blood monocytes and some of their subtypes, but these changes appear to be unrelated to blood serum factors.