The purpose of this study was to compare physical demands between game quarters and specific playing positions during official basketball competition. Thirteen professional male basketball players from the Spanish 2^nd Division were monitored across all 17 regular-season home games. Physical demands were analyzed using a local positioning system (WIMU PRO™, Realtrack Systems S.L., Almería, Spain) and included peak velocity, total distance covered, high-speed running (>18 km·h^-1), player load, jumps (>3G), impacts (>8G) and high-intensity accelerations (≥2 m·s^-2) and decelerations (≤-2 m·s^-2). A linear mixed model was used to test statistical significance (p < 0.05) between independent variables. Furthermore, standardized Cohen’s effect size (ES) and respective 90% confidence intervals were also calculated. There was an overall decrease in all variables between the first and fourth quarter during competition. Specifically, total distance covered (p < 0.001; ES = -1.31) and player load (p < 0.001; ES = -1.27) showed large effects between the first and last period. Regarding differences between positions, guards presented significant increased values compared to centers (p = 0.04; ES = 0.51), whereas centers achieved significant larger results and moderate effects in comparison to guards in peak velocity (p = 0.01; ES = 0.88) and jumps (p = 0.04; ES = 0.86). In conclusion, physical demands vary between game quarters and playing positions during official competition and these differences should be considered when designing training drills to optimize game performance.