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To investigate the aphrodisiac potential of polyherbal formulations prepared from different parts of Angelica sinensis, Aѕtragaluѕ membranaceuѕ, Cistanche tubulosa, Cynomorium songaricum, Epimedium sagittatum, Salvia miltiorrhiza, Paeoniae alba, and Peliosanthes micrantha in inexperienced male Sprague-Dawley rats. The different concentrations of prepared polyherbal formulations i.e. 430 and 860 mg/kg and testosterone injection positive control were administered orally to rats (n = 11 animals per group) for 7 days. The following sexual performances were evaluated: mount frequency (MF), mount latency (ML), intromission frequency (IF), intromission latency (IL), ejaculation frequency (EF), post-ejaculatory interval (PEI), ejaculation latency (EL); and two indexes such as copulatory efficiency (CE), intromission ratio (IR) for single dose and repeated doses of 7 days. Mating behavior parameters in male rats were monitored on days 1 and 7 of treatment pairing with receptive females. After termination of drug treatment, the body weights, weights of reproductive organs, and levels of testosterone were also evaluated. The present results over 7 days of the repeated dose experiment showed increases in body weights, up to 6.13% at a high dose of 860 mg/kgP, significantly increased weight gains in reproductive organs, and significantly increased levels of serum testosterone in treated rats. The tested prescription showed different effects of sexual behavior parameters between acute single dose and subacute repeated doses during 7 days of administration. In the acute study, the prescription improved the immediate performance of sexually inexperienced male rats. In the subacute research, the administration at high dose rats improved the mount frequency (MF) and ejaculation frequency (EF). On the contrary, the increase of mount latency (ML) and post-ejaculatory interval (PEI) might indicate long use of the formulation can decrease its ED treatment efficiency. In conclusion, the polyherbal formulation showed a significant increase in serum testosterone levels, weights of reproductive organs as well as mating behaviors as compared to controls, suggesting good aphrodisiac activity on male Sprague-Dawley rats in the above experimental model. However, the repeated use of polyherbal formulation is less effective in ED treatment.