Context: While a number of studies have researched road-cycling performance, few have attempted to investigate the physiological response in field conditions. Purpose: To describe the physiological and performance profile of an uphill time trial (TT) frequently used in cycling competitions. Methods: Fourteen elite road cyclists (mean ± SD age 25 ± 6 y, height 174 ± 4.2 cm, body mass 64.4 ± 6.1 kg, fat mass 7.48% ± 2.82%) performed a graded exercise test to exhaustion to determine maximal parameters. They then completed a field-based uphill TT in a 9.2-km first-category mountain pass with a 7.1% slope. Oxygen uptake (VO2), power output, heart rate (HR), lactate concentration, and perceived-exertion variables were measured throughout the field-based test. Results: During the uphill TT, mean power output and velocity were 302 ± 7 W (4.2 ± 0.1 W/kg) and 18.7 ± 1.6 km/h, respectively. Mean VO2 and HR were 61.6 ± 2.0 mL · kg−1 · min−1 and 178 ± 2 beats/min, respectively. Values were significantly affected by the 1st, 2nd, 6th, and final kilometers (P < .05). Lactate concentration and perceived exertion were 10.87 ± 1.12 mmol/L and 19.1 ± 0.1, respectively, at the end of the test, being significantly different from baseline measures. Conclusion: The studied uphill TT is performed at 90% of maximum HR and VO2 and 70% of maximum power output. To the authors’ knowledge, this is the first study assessing cardiorespiratory parameters combined with measures of performance, perceived exertion, and biochemical variables during a field-based uphill TT in elite cyclists.