Abstract Hematological and blood biochemical reference information is important to establish physiological status of freshwater stingray populations and improve care and management protocols in artificial environments. Here, we used a commercial freshwater stingray with high mortality rates in the market (Potamotrygon magdalenae), as an example to understand how artificial environments and handling protocols influence physiological status of captive freshwater stingrays. To this purpose, blood from five adult males and six adult females was collected to perform complete blood counts and blood chemistry analyses. All sampled animals showed good body condition with no differences between sexes. Differences between sexes were only found for the differential count of lymphocytes. Red blood results were consistent with previously studied potamotrygonids while white blood results showed higher values of leukocytes, thrombocytes, heterophils and lymphocytes in P. magdalenae compared to other Potamotrygonids. All types of leukocytes described for elasmobranchs were found except neutrophils and basophils. Blood metabolites showed an influence of ex situ diet in total protein, triglycerides and cholesterol. Glucose results were consistent while urea showed lower levels than those recorded for other freshwater stingrays. These results highlight the importance of physical, physiological and health analysis in freshwater stingrays as a part of welfare assessment to improve monitoring protocols and survival rates in public or private aquaria.