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Exercise has emerged as a fundamental therapeutic medicine in the management of cancer and is associated with a lower risk of recurrence and increased survival. A growing body of evidence has emerged on the acute effects of a single bout of exercise as well as chronic effects in suppressing growth of different cancer cell lines. The purpose of this review was to systematically examine the acute effects of exercise-conditioned serum and determine the impact of chronic exercise-conditioned serum on cancer cells in vitro. A systematic search was undertaken in PubMed, Medline, CINAHL, EMBASE, SPORTDiscus (via EBSCOhost), and Web of Science from inception to July 2024. Eligible studies examining the effects of acute and chronic exercise training on cancer cells in vitro were included. Fourteen studies met the eligibility criteria (n = 423). Acute exercise-conditioned serum has inhibitory effects on different cancer cell lines in vitro, when performed at moderate to high intensity, regardless of the training mode. For the chronic effects of exercise-conditioned serum the findings were mixed with some studies showing cancer-suppressive effects, while others reported no impact. Evidence suggests that acute exercise-conditioned serum can inhibit cancer cell growth in vitro, including breast, prostate, colorectal, and lung cancers. However, chronic effects are inconsistent, with some studies showing cancer-suppressive effects on breast and prostate cancer cells, while others show no change. Limitations of the studies should be considered, and additional research is necessary to determine the role of exercise prescription specifics such as mode and volume/intensity.