Increasing ecotourism activity in Argentine Patagonia has led to concerns about the effects of ecotourism on wildlife populations. Penguin breeding colonies are popular tourist destinations. While some species of penguins habituate to human visits, others exhibit negative effects due to disturbance. We studied the effects of tourism on Magellanic Penguins (Spheniscus magellanicus) at San Lorenzo colony located on the Peninsula Valdes, Chubut, Argentina. We measured behavioural, physiological, and reproductive parameters in birds nesting in tourist-visited and non-visited areas. San Lorenzo has 11 years of visitation history and a low intensity of visitors (~10,000 annually). After 15 min of a simulated tourist visit, penguins in the tourist area showed fewer alternate head turns and lower plasma levels of the stress hormone corticosterone than penguins in a non-visited area. However, penguins showed similar baseline levels of corticosterone between areas as well as similar levels of integrated corticosterone expressed after an acute stressor. Penguin breeding success and chick growth were similar between areas. We compared these findings to results previously published from Punta Tombo, a colony with a longer history (+50 years) and high intensity of annual visitors (>120,000 people). Many (reproductive and behavioural parameters), but not all (physiological parameters), of our findings are similar. That a physiological difference exists may suggest that the previous history of human visitation plays an important role in the response of the birds. Thus, the continuous monitoring of tourist activity is important, as a history of visitation disturbance seems to have an effect on how birds respond to tourists.