Background/Objectives: High calcium intakes enhance fat loss under restricted energy intake. Mechanisms explaining this may involve reduced dietary fat absorption, enhanced lipid utilization and (or) reductions in appetite. This study aimed to assess the impact of 2 weeks of calcium supplementation on substrate utilization during exercise and appetite sensations at rest. Subjects/Methods: Thirteen physically active males completed two 14-d supplemental periods, in a double-blind, randomized crossover design separated by a 4-week washout period. During supplementation, a test-drink was consumed daily containing 400 and 1400 mg of calcium during control (CON) and high-calcium (CAL) periods, respectively. Cycling-based exercise tests were conducted before and after each supplemental period to determine substrate utilization rates and circulating metabolic markers (non-esterified fatty acid, glycerol, glucose and lactate concentrations) across a range of exercise intensities. Visual analog scales were completed in the fasting, rested state to determine subjective appetite sensations. Results: No significant differences between supplements were observed in lipid or carbohydrate utilization rates, nor in circulating metabolic markers (both P>0.05). Maximum rates of lipid utilization were 0.47±0.05 and 0.44±0.05 g/min for CON and CAL, respectively, prior to supplementation and 0.44±0.05 and 0.42±0.05 g/min, respectively, post-supplementation (main effects of time, supplement and time x supplement interaction effect all P>0.05). Furthermore, no significant differences were detected in any subjective appetite sensations (all P>0.05). Conclusions: Two weeks of calcium supplementation does not influence substrate utilization during exercise in physically active males.