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Abstract Media supplemented with sera from acutely exercised men has been shown to have ‘anti‐cancer’ effects on prostate and breast cancer cell lines. This study investigated whether media supplemented with plasma samples taken at rest (≥30 h since the most recent exercise session) from men who were endurance‐trained (END), strength‐trained (STR) or recreationally active controls (CON) impacted the results of four assays that mimic hallmarks of cancer (proliferation, migration, extracellular matrix invasion and anoikis resistance) in the BT‐549 breast cancer cell line. Compared to control conditions of either serum‐free media or fetal bovine serum as appropriate, BT‐549 cells cultured with plasma‐supplemented media regardless of group resulted in greater cell proliferation (∼20–50%) and cell migration (∼15–20%), and lower extracellular matrix invasion (∼10–20%) and anoikis resistance (∼15–20%). Supplementing media with plasma from END or STR did not impact any outcomes of these assays compared to plasma from CON. Media supplemented with human plasma can impact functional assays reflective of cancer hallmarks in BT‐549 cells, but effects of exercise on proliferation, migration, extracellular matrix invasion and anoikis resistance were not evident in resting blood samples of individuals with a prior history of exercise training.