Background: Caffeine ingestion improves athletic performance, but impairs sleep quality. We aimed to analyze the effect of caffeine intake on 800-m running performance, sleep quality (SQ), and nocturnal cardiac autonomic activity (CAA) in trained runners. Methods: Fifteen male middle-distance runners participated in the study (aged 23.7 ± 8.2 years). In a randomized and comparative crossover study design, the athletes ingested a placebo (PL) or caffeine supplement (CAF; 6 mg∙kg−1) one hour before an 800-m running time-trial test in the evening. During the night, CAA and SQ were assessed using actigraphy and a sleep questionnaire. A second 800-m running test was performed 24 h after the first. Time, heart rate, rating of perceived exertion, and blood lactate concentration were analyzed for each running test. Results: No significant differences in CAA and performance variables were found between the two conditions. However, CAF impaired sleep efficiency (p = 0.003), actual wake time (p = 0.001), and the number of awakenings (p = 0.005), as measured by actigraphy. Also, CAF impaired the questionnaire variables of SQ (p = 0.005), calm sleep (p = 0.005), ease of falling asleep (p = 0.003), and feeling refreshed after waking (p = 0.006). Conclusion: The supplementation with caffeine (6 mg∙kg−1) did not improve the 800-m running performance, but did impair the SQ of trained runners.