West Indian manatees (Trichechus manatus) are captured, handled, and transported to facilitate conservation, research, and rehabilitation efforts. Monitoring manatee oral temperature (OT), heart rate (HR), and respiration rate (RR) during out-of-water handling can assist efforts to maintain animal well-being and improve medical response to evidence of declining health. To determine effects of capture on manatee vital signs, we monitored OT, HR, and RR continuously for a 50-min period in 38 healthy, awake, juvenile and adult Florida manatees (T. m. latirostris) and 48 similar Antillean manatees (T. m. manatus). We examined creatine kinase (CK), potassium (K+), serum amyloid A (SAA), and lactate values for each animal to assess possible systemic inflammation and muscular trauma. OT range was 29.5 to 36.2° C, HR range was 32 to 88 beats/min, and RR range was 0 to 17 breaths/5 min. Antillean manatees had higher initial OT, HR, and RR than Florida manatees (p < 0.001). As monitoring time progressed, mean differences between the subspecies were no longer significant. High RR over monitoring time was associated with high lactate concentration. Antillean manatees had higher overall lactate values ([mean ± SD] 20.6 ± 7.8 mmol/L) than Florida manatees (13.7 ± 6.7 mmol/L; p < 0.001). We recommend monitoring manatee OT, HR, and RR during capture and handling in the field or in a captive care setting.