This study aimed to determine the effect of a short-graded with respect to a long-graded protocol laboratory test on the physiological responses of road cyclists. Twenty well-trained road cyclists performed a short-graded and long-graded laboratory tests within 1 week of each other in a randomized and crossover study design. Blood lactate concentration ([La-]b), heart rate (HR), oxygen consumption ((Equation is included in full-text article.)), and carbon dioxide production ((Equation is included in full-text article.)) were measured. Fat and carbohydrate oxidation rates (FAT(OxR) and CHO(OxR)) were estimated at the end of each stage during the short-graded and the long-graded (10th minute: T2.10) and in the middle of long-graded (fifth minute: T2.5) protocol. Lactate threshold (LT) and individual anaerobic threshold (IAT) were calculated. For maximal intensities, duration and maxFAT(OxR) were significantly higher in long-graded with respect to short-graded protocols. Peak power output (POPeak), HRPeak, [La-]bmax, (Equation is included in full-text article.), and maxCHO(OxR) were significantly higher in short-graded with respect to long-graded protocols. At submaximal intensities, short-graded protocol provoked higher demands on glycolytic metabolism than long-graded protocol; no differences were illustrated for HR or (Equation is included in full-text article.)between protocols. Crossover concept shifted to higher intensities in long-graded with respect to short-graded protocols due to the higher lipolytic response during the long-graded protocol. Both LT and IAT were reached at the same %(Equation is included in full-text article.), although significantly higher PO in short-graded with respect to long-graded protocols was reached. The long-graded proved to be more specific than the short-graded protocol to assess the physiological responses of road cyclists based on relative PO (W·kg(-1)).