This paper reports on studies testing the hypothesis that partially oxidized biodiesel fuel remaining in a vehicle fuel tank could accelerate the degradation of fresh biodiesel after refueling. A blend of soybean oil fatty acid methyl esters (FAME) in petroleum diesel fuel (30% v:v biodiesel, B30) was aged under accelerated conditions (90°C, 100 mL/min aeration). Aging conditions focused on producing large amounts of oxidation products in three scenarios: 1) significantly reduced oxygen stability (Rancimat induction period); 2) high peroxide values (PV); and 3) considerable formation of acids as measured by total acid number (TAN). Aged B30 fuel was added to fresh B30 fuel at two concentrations (10% and 30% v/v aged fuel) and oxidation of the mixtures at the above aging conditions was monitored for Rancimat induction period, PV, TAN, and FAME composition. The higher content of aged fuel carryover (30% v/v) 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 not greatly affected by the different scenarios. These results provide greater insight into potential issues associated with biodiesel fuel aging under high-temperature diesel fuel system conditions.