Background. Blinded clinical trials have reported a modest and transient "start-up syndrome" with initiation of tenofovir-based pre-exposure prophylaxis (PrEP). We evaluate this phenomenon and its effect on adherence in an open-label PrEP study. Methods. In the iPrEx open-label extension (OLE) study, an 18-month open-label, multi-site PrEP cohort taking daily oral coformulated tenofovir/emtricitabine, we examined the prevalence and duration of PrEP-associated symptoms and their effect on adherence, assessed by drug levels in dried blood spots tested monthly for the first 3 months. Results. Symptom reports peaked within the first month, with 39% reporting potentially PrEP-related symptoms compared to 22% at baseline. Symptoms largely resolved to pre-PrEP levels by 3 months. Symptoms varied substantially in frequency by study site (range in 1-month symptoms: 11% to 70%). Nongastrointestinal (GI) symptoms were not associated with adherence (odds ratio OR = 1.2, 95% confidence interval CI, .4–3.7); however, GI-associated symptoms in the first 4 weeks were inversely associated with adherence at 4 weeks (OR = 0.47, 95% CI, .23–.96). Reports of GI symptoms were associated with 7% (95% CI, 4%–11%) of suboptimal adherence in this cohort. Conclusions. PrEP-associated symptoms in the open-label setting occur in a minority of users and largely resolve within 3 months. GI symptoms are associated with a modest reduction in PrEP adherence, but good adherence is possible even in the presence of frequent symptom reports. Clinical Trials Registration. Clinicaltrials.gov NCT00458393.