Endurance athletes are at increased risk of relative energy deficiency associated with metabolic perturbation and impaired health. We aimed to estimate and compare within-day energy balance (WDEB) in male athletes with suppressed and normal resting metabolic rate (RMR) and explore if within-day energy deficiency (WDED) is associated with endocrine markers of energy deficiency. Thirty-one male cyclists, triathletes, and long-distance runners recruited from regional competitive sports clubs were included. The protocol comprised measurements of RMR by ventilated hood, and energy intake and energy expenditure to predict RMRratio(measured RMR/predicted RMR), energy availability (EA), 24-hour energy balance (EB) and WDEB in 1-hour intervals, assessment of body-composition by dual-energy X-ray absorptiometry, and blood plasma analysis. Subjects were categorized as having suppressed (RMRratio< 0.90, n=20) or normal RMR (RMRratio> 0.90, n=11). Despite no observed differences in 24-hour EB or EA between the groups, subjects with suppressed RMR spent more time in an energy deficit exceeding 400 kcal (20.9 [18.8 - 21.8] hours vs. 10.8 [2.5 - 16.4], P=0.023), and had larger single-hour energy deficits compared to subjects with normal RMR (3265 ± 1963 kcal vs. -1340 ± 2439, P=0.023). Larger single-hour energy deficits were associated with higher cortisol levels (r = -0.499, P=0.004) and a lower testosterone:cortisol ratio (r = 0.431, P=0.015), but no associations with T3or fasting blood glucose were observed. In conclusion, WDED was associated with suppressed RMR and catabolic markers in male endurance athletes.