This study was conducted (1) to evaluate the effects of photoperiod (fixed vs. decreasing light), fish size (136 vs. 220 mm), dissolved ions (hardness and salinity) and diet (menhaden oil vs. coconut oil-based) on the tolerance (survival) of Nile tilapia, Oreochromis niloticus, to low temperatures (decreased by approximately 0.5 °C per day) and (2) to evaluate the effect of dietary fatty acid composition on selected physiological characteristics of Nile tilapia exposed to decreasing temperatures. Size significantly affected mortality, with smaller fish being less tolerant to low temperatures than larger fish. Results were equivocal in the photoperiod, dissolved ion and dietary lipid experiments, and were dependent on the method of data analysis employed. Diet significantly affected plasma osmolarity, with higher values in fish fed the menhaden-based diet. Haematocrit, serum glucose, sodium and cortisol concentrations, serum and splenic lysozyme activities, lymphocyte count and hepatosomatic index were not affected by diet. Haematocrit, white cell count and serum glucose and sodium concentrations were significantly affected by temperature, but serum and splenic lysozyme content, hepatosomatic index, and serum cortisol concentrations were not. The results of this series of experiments indicate that altering the environment or diet has little effect on the ability of Nile tilapia to survive low temperatures.