Erythema migrans is the most common clinical manifestation of Lyme disease, with concomitant subjective symptoms occurring in ∼65% of cases in the USA. We evaluated the impact on 12 particular symptoms of having been started on antibiotic treatment before enrollment for 38 subjects with erythema migrans versus 52 untreated subjects. There were no significant differences in the frequency of having at least one symptom or in the symptom severity score on study entry. However, the frequency of having at least one symptom was significantly greater for those who had received <7 days of antibiotic treatment compared with those who had been treated for ≥7 days (23/24 [95.8%] vs 8/14 [57.1%], p=0.006). In addition, the percentage who were males was significantly lower among the group on treatment compared with the untreated study subjects (13/38 [34.2%] vs 34/52 [65.4%], p=0.005). In conclusion, based on these findings, combining untreated and on-treatment groups of patients with erythema migrans for research study analyses may have limitations and, depending on the study objectives, might not be preferred. Additional studies are warranted to better understand the day-to-day impact of antibiotic treatment on the presence, type, and severity of symptoms in patients with early Lyme disease.