Limited specificity of the tuberculin skin test incited the development of the intradermal Mycobacterium tuberculosis-specific rdESAT-6 skin test. Animal studies have shown, however, that there is a possible risk of sensitization when repeated injections of rdESAT-6 are given. The aim of this phase 1 open clinical trial was to assess the sensitization risk and safety of repeated administration of rdESAT-6 reagent in 31 healthy adult volunteers. Three groups of volunteers received two fixed doses of 0.1μg rdESAT-6 28, 56 or 112days apart, respectively. After the second injection, the diameter of induration and/or redness at the injection site was measured and taken as a possible sensitization reaction if >5mm. In vitro interferon γ (IFN-γ) responses were measured as supportive evidence. Local adverse reactions at the injection site and adverse events were recorded. One out of 31 (3%) volunteers showed a positive skin reaction (sensitization) upon a second injection of rdESAT-6 after 28days and an increased IFN-γ response to ESAT-6. For 7 (23%) of the volunteers, local adverse reactions related to the product were registered, but all reactions were mild and predictable. In conclusion, repeated injections of the rdESAT-6 skin test reagent are safe, and sensitization occurs at a low rate, especially if the time span between succeeding doses is wide.