Objective To determine the frequency and risk factors of post-dural puncture headache (PDPH) in research volunteers. Background Despite increasing interest in measuring cerebrospinal fluid biomarkers to investigate disease pathogenesis and diagnosis, previous case series have evaluated lumbar puncture (LP) safety only in clinical care. PDPH is a common complication after LP. Methods We determined the frequency of post-dural puncture headache (PDPH) in neurologically unselected HIV seropositive and seronegative adults volunteering for research, as well as the variables associated with the development of (PDPH). Variables studied were BMI, HIV serostatus, volume of CSF removed, number of previous LPs, use of pre-medication, LP position, lumbar space, number of needle passes, whether or not aspiration was used, CSF WBC, CSF RBC, CD4 count, CD4 nadir, CSF HIV viral load, plasma HIV viral load, and race. Results Of 675 LPs performed over one year, headache developed in 38 (5.6%; 95% CI 4.2, 7.1)). Most PDPH (92%) resolved spontaneously or with conservative medical management; 3 required epidural blood patch. Greater headache risk was associated with lower body mass index (BMI ≤ 25 versus > 25) (OR 3.3; CI 95% 1.5, 7.0; p=0.001) and less prior LP experience (previous LPs ≤ 2 vs > 2) (OR 2.1; CI 95% 1.1, 4.1; p=0.03). PDPH was not significantly (p > .05) related to HIV serostatus, CSF volume, or gender. Conclusion In this study, where tolerance to risk was low because LPs were done for research rather than clinical purposes and healthy controls were included, adverse effects were mild and self-limited.