Spermatozoa can only achieve its ultimate goal of fertilizing an oocyte if multiple sperm functions occur prior to, during and after fertilization. Semen samples, however, consist of a heterogeneous population of spermatozoa of which many are unable to reach or fertilize an oocyte. Sperm functional tests assess independent sperm functions and indirectly determine a male’s fertility status. One sperm functional aspect that is not currently assessed clinically is hyperactivation (HA). Sperm hyperactivated motility is imperative for both detachment from the oviductal wall and for penetration of the zona pellucida, subsequently resulting in fertilization. The main aim of this study was to investigate motility characteristics of human sperm subpopulations (SSP) and to induce HA with caffeine and procaine. Three SSP (good, medium and poor motility) were extracted from semen samples using PureSperm density gradient centrifugation. Sperm motility of SSP was assessed using computer-aided sperm analysis (CASA). Subsamples of SPP were exposed to 2mM caffeine and 5mM procaine for 15, 30, 60, 90 and 120 minutes. Percentage sperm displaying hyperactive motility was determined using cut-offs for curvilinear velocity, linearity and amplitude of lateral head displacement. The results indicate significant differences in overall motility, sperm kinematic parameters as well as HA among the three SSP (P<0.05). Procaine and caffeine both induced HA, although the most pronounced effect of procaine is evident after 15-30 minutes compared to caffeine (60-90 minutes). The existence and motility characteristics of SPP should be considered in future during clinical assessment of male fertility, especially when assessing HA.  The results from this study will be used to develop a sperm functional test for HA.