Aims: Assessing the reliability of electrical pain threshold (PT) and evaluating the impact of stimulation frequency at different locations in the healthy lower urinary tract (LUT). Methods: Ninety subjects (age: 18.3-35.8 years, 40 females, 50 males) were randomly allocated to one stimulation site (bladder dome [BD], trigone, proximal, membranous (only males), or distal urethra). Using 0.5 Hz/1.1 Hz/ 1.6 Hz electrical stimulation (square wave, pulse width:1 ms), current perception thresholds (CPTs), PTs and tolerance thresholds were assessed at two visits. Analyses were performed using linear mixed models, intraclass correlation coefficients (ICC) and Bland-Altman method.Results: PTs vary in relation to stimulation frequency, location and between genders. PT decreased with higher stimulation frequency. The highest PTs were measured at the BD and membranous urethra with males reporting higher PTs than females. Reliability of PT assessments according to ICC was good to excellent across all frequencies, locations, and genders (ICC = 0.61-0.97), except for BD and distal urethra in females showing poor to fair reliability (BD: all frequencies, distal urethra: 0.5 Hz).Conclusions: PTs can be safely and reliably assessed from bladder and urethral locations. Semi-objective PT assessment may provide additional information on functionality and sensitivity of the LUT slow fiber afferents and complement findings from urodynamic investigations and CPT assessments. In conclusion, the developed methodology may open new opportunities for using electrical stimulation paradigms for LUT PT assessments and diagnostics. All this allows a more precise, location-specific characterization of pain origin and pain reaction towards therapy. K E Y W O R D S afferent nerve fibers, electrical stimulation, humans, lower urinary tract, neurophysiology, pain threshold Stéphanie van der Lely and Martina D. Liechti shared the firstauthorship.