Fishways should attenuate the blockage of migratory routes imposed by dams. However, when fish ladders are poorly designed, they may cause excessive stress. This study evaluated if fish ladder transposition was a stressful event for Prochilodus lineatus in the Porto Primavera Dam, Paraná River, Brazil. Were analyzed the body weight, standard length and plasma concentrations of cortisol, glucose, lactate, chloride and hematocrit in 48 adult fish sampled at downstream, into the fish ladder and upstream. Data were analyzed by two-way ANOVA and Fisher–LSD post-hoc test. Correlation between lactate concentrations and, fish weight and standard length was determined using Pearson correlation. The significance level was 5%. Females were heavier and longer than males (F1, 42 = 20.47; P = 0.001), and both sex collected upstream of the dam also had a greater weight (F2, 42 = 5.05; P = 0.011). While cortisol and chloride concentrations did not vary, the both glucose concentrations increased significantly in the ladder compared to downstream (P < 0.001) but did not varied between fish from downstream and upstream (P = 0.31). Female glucose values were higher than those of males (F1, 42 = 5.30; P = 0.026). There was an increase in lactate concentration in the ladder (F2, 42 = 3.81; P = 0.03) and a negative correlation between lactate concentration and body weight (P = 0.027; ρ = -0.548) and between lactate concentration and standard length (P = 0.011; ρ = -0.613). Hematocrit values decreased in the ladder (F2, 42 = 4.66; P = 0.015). These results show that although the intense physical exertion of the fish in ladder transposition demanded high metabolic requirements and activated anaerobic pathways, it was not a stressful condition. We propose that physiological indicators should be used as criteria in projects for fishways, as well as post-construing assessment.