This study aimed to establish comprehensive reference values for horses working in developing countries, and to compare them against accepted values for horses in developed countries, supporting diagnosis and clinical decision-making. Horses in developing countries usually perform strenuous work in hot, resource-limited conditions, so their 'normal' blood parameters may differ from other horses. Blood was analysed from 203 working horses in Pakistan, meeting defined clinical criteria. Age, sex, body condition and work-type showed small significant effects, but none were clinically relevant. Of the 32 reference intervals, 28 overlapped those of UK horses. However, the entire reference interval for creatine kinase was higher than for UK horses, while those for erythrocytes, albumin and albumin:globulin ratio were lower. Haematocrit and haemoglobin concentrations were also low. Therefore, apparently healthy working horses may have chronic muscle damage from overwork, and may have sub-clinical anaemia. Interventions combating these conditions could improve animal welfare, although it is unclear whether differences between UK and Pakistan reference values reflect chronic abnormalities, or are in fact physiological adaptations enabling horses to cope with the challenging conditions.