Handwashing practices may be adversely influenced by the detrimental effects of handwashing on skin. A protocol was developed to assess the physiologic and microbiologic effects of frequent handwashing. Fifty-two female volunteers washed their hands 24 times per day for 5 days. Five agents were tested: water alone, non-medicated bar soap, a chlorhexidine-containing antiseptic, and two agents containing povidone-iodine (one currently available on the market and one being tested for possible marketing). Some damage to the outer membrane of skin, the stratum corneum, occurred in all groups. There were significant changes in the amount of evaporation water loss (p = .001) and in self assessments of skin condition (p = .005) from pre-to-post test for the entire group. Skin damage was also assessed by visualizing desquamating stratum corneum cells, which are shed in large aggregates when detergents injure skin. Significantly less such shedding occurred in subjects using water alone, bar soap, and the chlorhexidine formulation (p = .02). Greater antimicrobial activity of an agent was not correlated with increased skin trauma. We have quantitated, using objective physiologic parameter, the skin damage that occurs during even a short period of frequent handwashing. We recommend that further studies using the methods described be conducted to quantitate skin damage over longer periods of time, more closely resembling handwashing practices of health care personnel.