Human spermatozoa interact with a complex biochemical environment in the female reproductive tract en route to the site of fertilisation. Ovarian follicular fluid contributes to this complex milieu and is known to contain steroids such as progesterone, whose effects on sperm physiology have been widely characterised. We have previously reported that progesterone stimulates intracellular calcium concentration ([Ca2+]i) signalling and acrosome reaction in human spermatozoa. To characterise the effects of the unified complete follicular fluid steroid hormone complement on human spermatozoa, a comprehensive, data-based, 'physiological standard' steroid hormone balance of follicular fluid (shFF) was created from individual constituents. shFF induced a rapid biphasic [Ca2+]i elevation in human spermatozoa. Using population fluorimetry, we compared [Ca2+]i signal amplitude in cells exposed to serial applications of shFF (6 steps from 10-5X up to 1X shFF) with responses to the equivalent progesterone component alone (6 steps from 135 pM - 13.5μM). Threshold for the response to shFF was right-shifted (≈10-fold) compared to progesterone alone, but the maximum response to shFF was greatly enhanced. An acrosome reaction assay was used to assess functional effects of shFF-induced sperm calcium signalling. shFF as well as progesterone-treated spermatozoa showed a significant increase in % acrosome reaction (P < 0.01). All of this evidence suggests the modulation of progesterone-mediated responses by other follicular fluid steroids.