Foliar sugar accumulation in grapevines with leafroll disease was correlated with lower photosynthesis, likely due to feedback inhibition. However, cold acclimation of dormant tissues remained unaffected by the virus status. Grapevine leafroll-associated viruses (GLRaV) contribute to losses in fruit yield and quality worldwide. Visually, leafroll disease symptoms appear similar to those associated with an imbalance in source/sink relations and a concomitant feedback inhibition of photosynthesis, which is often caused by an impasse in sugar translocation. In order to test this potential relationship and related physiological responses, leaf water status, gas exchange, non-structural carbohydrates, and dormant tissue cold hardiness were examined over 2 years in healthy and GLRaV-3-infected, field-grown Merlot grapevines. Diurnal and seasonal changes in leaf water status and gas exchange were dominated by variations in water availability, temperature, and leaf age, while GLRaV-3 infection contributed less to the overall variation. By contrast, foliar carbohydrates increased markedly in infected plants, with starch accumulating early in the growing season, followed by soluble sugar accumulation, leaf reddening, and declining gas exchange. Photosynthesis correlated negatively with leaf sugar content. However, dormant-season cold hardiness of buds and cane vascular tissues was similar in healthy and infected vines. These findings support the idea that visible symptoms of grapevine leafroll disease are a consequence of carbohydrate accumulation which, in turn, may lead to feedback inhibition of photosynthesis. In addition, this study provided evidence that GLRaV-3 infection is unlikely to alter the susceptibility to moderate water deficit and winter damage in mature Merlot grapevines.