In Australia, temperatures below 11°C (called cold shocks) were believed to retard cotton (Gossypium hirsutum L.) growth, development and yield. Recent studies, however, have suggested that temperatures lower than this did not impede cotton development beyond normal developmental responses to cool temperatures. This paper aims to test the hypothesis that cold exposure to 10°C for 10 and 20 nights does not reduce tissue viability in vegetative and early flowering cotton plants. Cold temperatures at 10°C for 10 and 20 consecutive nights were imposed on cotton plants, grown in both controlled temperature glasshouses and outdoors, at the vegetative seedling and early flowering stages. Extreme temperature tests at 2, 5 and 7°C for two nights were also imposed to generate tissue damage for comparison. 2,3,5-Triphenyl tetrazolium chloride (TTC) tissue viability (testing for mitochondrial activity), relative electrical conductivity (REC, testing for membrane integrity), leaf chlorophyll fluorescence, leaf photosynthesis, plant dry weight and yield were measured. Only exposure at 2°C for two nights showed negative effects in the TTC and REC tests, and leaves of these plants died soon after exposure. There were no consistent negative effects in the TTC and REC tests for all treatments at 10°C for 10 and 20 nights compared with the respective controls, suggesting that there was no structural or functional damage to leaves. In support of these findings, leaf photosynthesis and both light- and dark-adapted chlorophyll fluorescence for the 20 nights at 10°C treatment were occasionally below the controls but recovered quickly, suggesting that only temporary dynamic photoinhibition occurred. Cotton plant development was delayed following 10 and 20 nights at 10°C owing to reduced degree day accumulation. These data support previous work that cold temperatures at 10°C for up to 20 nights would be unlikely to result in yield reduction as a consequence of plant damage, and also that crop development can be estimated with degree days without an adjustment for cold shock. The use of TTC and REC has potential for novel detection of tissue damage for cotton at extreme temperatures.