Traditional endpoints of the double-ended test (DET), a contraceptive screening assay used to evaluate the ability of a compound to permeate cervical mucus and inhibit sperm progression, ignore important information about sublethal effects upon sperm cells. Improved contraceptive agents may capitalize on such sublethal aspects. This study utilized a DET testing protocol that included measurement of human sperm motion characteristics as an indicator of cell function within spermicide-exposed human mucus. The currently available spermicide nonoxynol-9 (N9) was used as the test compound and was dissolved in two different delivery solutions, deionized (DI) water and saline, to evaluate the effects of the osmolarity and pH of the delivery vehicle on test results. The N9-water treatment demonstrated significantly greater activity than the N9-saline treatment in terms of all measured variables, exhibiting an apparent "biopermeation" distance approximately 3 mm further into the mucus. The DI water control treatment displayed less activity than N9-saline in terms of the vanguard penetration distance, but comparable or greater activity in terms of inhibiting kinematic variables. The saline control treatment had no effect in terms of any measured variable. Dose responses to N9 of sperm in mucus were inferred from DET results combined with direct measures of N9 diffusion. These were compared to dose responses to N9 of seminal sperm, indicating that N9 inhibits sperm motion at lower concentrations in mucus than in semen.