A common feature of human aging is the acquisition of somatic mutations, and mitochondria are particularly prone to mutation due to their inefficient DNA repair and close proximity to reactive oxygen species, leading to a state of mitochondrial DNA heteroplasmy1,2. Cross-sectional studies have demonstrated that detection of heteroplasmy increases with participant age3, a phenomenon that has been attributed to genetic drift4-7. In this first large-scale longitudinal study, we measured heteroplasmy in two prospective cohorts (combined n=1405) at two timepoints (mean time between visits, 8.6 years), demonstrating that deleterious heteroplasmies were more likely to increase in variant allele fraction (VAF). We further demonstrated that increase in VAF was associated with increased risk of overall mortality. These results challenge the claim that somatic mtDNA mutations arise mainly due to genetic drift, instead demonstrating positive selection for predicted deleterious mutations at the cellular level, despite an negative impact on overall mortality.