The challenge hypothesis posits that acute increases in testosterone (T) during male-male competition enhance performance and survivability while limiting the physiological costs of consistently high T. Human challenge hypothesis research focuses on young men in industrial populations, who have higher baseline T levels than men in subsistence populations. We tested whether the Tsimane, pathogenically stressed forager-horticulturalists of the Bolivian Amazon, would express acute T increases in response to physical competition. Saliva was collected from 88 Tsimane men (aged 16-59 years) before and after a competitive soccer match. Tsimane men had significantly lower baseline levels of T (b ¼ 20.41, p , 0.001) when compared with age-matched United States (US) males. Linear mixed-effects models were used to establish that T increased significantly immediately following competition (b ¼ 0.23, p , 0.001), remaining high 1 h later (b ¼ 0.09, p ¼ 0.007); equivalent to 30.1 and 15.5 per cent increases in T, respectively. We did not find larger increases in T among winners (p ¼ 0.412), although T increases were positively associated with self-rated performance (b ¼ 9.07, p ¼ 0.004). These results suggest that despite lower levels of T than US males, Tsimane males exhibit acute increases in Tat the same relative magnitude reported by studies in industrialized settings, with larger increases in T for those who report better individual performance.