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Introduction: Coronavirus disease 2019 (COVID-19) has increased the frequency of handwashing. There is scarce evidence regarding the impact of different hand hygiene procedures on skin barrier function in clinical practice., Objective: To compare the impact on skin barrier function of different hand hygiene measures in healthcare workers in daily practice., Methods: A randomized controlled clinical trial was conducted. Participants were randomized to sanitize their hands with water and soap, alcohol-based hand sanitizers (ABHSs), or disinfectant wipes during their 8-hour working shift. Epidermal barrier functional parameters, such as transepidermal water loss (TEWL), and the microbial load were assessed before and immediately after the working day. Tolerance and acceptability of each product were recorded after work., Results: Sixty-two participants were included and 20, 21, and 21 were randomized to use water and soap, ABHS, and disinfectant wipes, respectively. After the 8-hour shift, TEWL increase was higher with disinfectant wipes than with soaps or ABHS (+5.45 vs +3.87 vs -1.46 g h -1  m -2 , respectively; P = .023). Bacteria and fungi colony-forming unit (CFU) count reductions were lower for the water and soap group than for ABHS and disinfectant wipes. Disinfectant wipes were considered more difficult to use (P = .013) compared with water and soap and ABHS., Conclusion: Daily hand hygiene with ABHS showed the lowest rates of skin barrier disruption and the highest reduction of CFU., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)