Background: There is scarce information detailing clinical and physiological effects of reversible injectable protocols of chemical restraint on Neotropical primates. Methods: Nineteen captive Spix´s Owl monkeys (Aotus vociferans) were assessed in a double-blind randomized crossover study using the following: ketamine/xylazine [KX], ketamine/midazolam [KM] and ketamine/xylazine/midazolam [KXM]. During immobilization, respiratory and pulse rates, rectal temperature, haemoglobin oxygen saturation and arterial blood pressure were recorded at 5-minute intervals during a 20-minute period; afterwards, antagonist drugs (yohimbine for xylazine and flumazenil for midazolam) were, respectively, administered. Quality and duration of induction, immobilization and recovery periods were recorded. Results: Ketamine/xylazine increased manipulation sensitivity and produced poor muscle relaxation. KM maintained all assessed parameters within physiological ranges. KXM produced depressant cardiorespiratory effects and hypotension. All protocols produced hypothermia. Conclusions: Based on its adequate anaesthetic depth and minimum effects on physiological parameters, KM is suitable for immobilizing A vociferans and performing short-term procedures lasting around 20 minutes.