This study investigates the effects of pharmacological cholinergic manipulation and aging on heart rate variability (HRV) and performance during an odor discrimination task in Long-Evans rats. Rats from distinct age cohorts (“Adult,” “Older Adult,” and “Elderly”) completed proactive interference (PI) odor-detection tasks using an olfactometer. Proactive interference refers to the phenomenon where previously learned information impedes the acquisition of new, related material. Pharmacological interventions involved systemic intraperitoneal (IP) injections of normal saline (0.9% sterile medical grade), methylscopolamine (0.25 mg/kg), or scopolamine (0.25 mg/kg). The ability to overcome PI was evaluated for each injection type. HRV data were collected via surgically implanted telemeters recording electrocardiogram (ECG) data. Results revealed no significant age-related differences in HRV between the “Elderly” and “Adult” cohorts. Pharmacological manipulation affected PI resolution differently between age cohorts, with adult rats showing impaired PI resolution under scopolamine and methylscopolamine compared to saline controls, while older adult rats behaved comparably across each injection type. These data suggest that cholinergic drugs may affect the body differently in naturally aged subjects. Unexpectedly, rats also experienced declines in baseline and new learning accuracy from cholinergic manipulation, which may be due to learning odor discriminations at an older age compared to previous studies that used younger animals. Rats experienced a drop in HRV after injection, suggesting a reduction in vagal tone due to reduced cardiac parasympathetic regulation.