An experimental study of the effects of partially-oxidized biodiesel fuel on the degradation of fresh fuel was performed. A blend of soybean oil fatty acid methyl esters (FAMEs) in petroleum diesel fuel (30% v:v biodiesel, B30) was aged under accelerated conditions (90°C with aeration). Aging conditions focused on three different degrees of initial oxidation: 1) reduced oxidation stability (Rancimat induction period, IP); 2) high peroxide values (PV); and 3) high total acid number (TAN). Aged B30 fuel was mixed with fresh B30 fuel at two concentrations (10% and 30% m:m) and degradation of the mixtures at the above aging conditions was monitored for IP, PV, TAN, and FAME composition. Greater content of aged fuel carryover (30% m:m) corresponded to stronger effects. Oxidation stability was most adversely affected by high peroxide concentration (Scenario 2), while peroxide content was most reduced for the high TAN scenario (Scenario 3). However, changes in TAN and FAME composition were modest with all four scenarios reaching a plateau in TAN formation at similar times and FAME concentrations showing similar declines. The results are discussed with respect to the chemistry of biodiesel fuel aging under high-temperature diesel fuel system conditions and considerations associated with the mixing of aged fuels with fresh fuels following vehicle refueling.