Environmental factors might influence the carbon balance and sugar content in grapevine. In this two-year research, the STELLA software was employed to predict dry matter accumulation in Sangiovese vines, comparing the traditional vertical shoot positioning (VSP) and the single high wire (SHW) trellis systems. Every week, vegetative, eco-physiological and grape quality parameters were collected for 15 tagged vines per trellis system to set up the software. Significant differences in photosynthesis were recorded in 2014, with higher values in VSP (23–25% more). Shoot growth was significantly higher in VSP (20–25% more), whereas higher dry matter (30%) and yield (9–11% more) were detected for SHW. At harvest, berry composition suggested a slower ripening in SHW compared to VSP, which was linked to the shading of clusters in SHW. Finally, for the first time, linear regressions were found between measured berry sugar content and STELLA-estimated dry matter (R2 = 0.96 in VSP; R2 = 0.95 in SHW). This latter evidence allowed the estimation of berry sugar content, showing this software to be a practical tool to support winegrowers in decision making. Other studies are already underway to calibrate and validate the model for other varieties, training systems and environments.