This application, in response to RFA-CA-004 “Research Answers to National Cancer Institute's (NCI) Provocative Questions (R01 Clinical Trial Optional),” will address “PQ2: How does intermittent fasting affect cancer incidence, treatment response, or outcome?” Obesity and age are two major risk factors for cancer development. Thus, therapeutic interventions that prevent or delay the development of excessive weight gain and/or age-associated physiological dysfunction hold great promise for reducing cancer risk in the increasingly obese and elderly global population. One such intervention is time-restricted eating (TRE), a pragmatic form of intermittent fasting in which daily caloric intake is constrained to a consistent window of 8–12 hours without explicitly reducing total caloric intake. In young male mice, time-restricted feeding (TRF) reduces cancer risk by preventing obesity and metabolic diseases. TRF has also been shown to reduce breast cancer xenograft progression in obese mice. In humans, short-term clinical studies of TRE have revealed metabolic improvements that predict reduced cancer risk, and epidemiological evidence suggests that prolonged nightly fasting can reduce the risk of cancer, independent of changes in body weight. This promising preliminary evidence suggests that TRE may be an effective intervention for reducing cancer risk. However, the effects of TRF in aged animals and in the context of an obesogenic Western diet have not yet been established, and the mechanisms by which TRF reduces cancer risk remain unknown. This application builds upon promising preliminary data and leverages the complementary skills of the research team to address these critical gaps in knowledge. Both obesity and aging are associated with mitochondrial dysfunction and the production of pro-tumorigenic mitochondrial metabolites. Proposed experiments test the hypothesis that TRF optimizes mitochondria function through both cell-autonomous and systemic mechanisms, thereby reducing cancer risk. In Aim 1, the impact of TRF on mitochondria function and related physiologies will be established in aged mice. Nutrient metabolism, energy consumption, and mitochondria function will be assessed in these mice. In Aim 2, an innovative combination of metabolomics and mitochondria respiration assays will be used to test the impact of TRF on mitochondria function in normal and cancer cells (assessing both cell-autonomous and non-cell-autonomous mechanisms). The effects of TRF on tumor incidence will be assessed by subjecting tumor-prone mice to TRF. In Aim 3, plasma collected from a recently concluded human TRE intervention study will be used to test the effect of TRE on mitochondria function and cancer risk in humans. The proposed comparative analysis of TRE in humans and mice will provide critical mechanistic insight into how one form of intermittent fasting can help prevent cancer onset and improve treatment outcomes.