Estimation of urine output and purine derivatives (PD) excretion from spot samples is possible because daily creatinine excretion is proportional to animal BW. Feeding regimen was so far omitted in the evaluation of effects on this estimation, and this study aimed to explore the effect of ad libitum or restricted intakes on estimation in spot urinary sampling. Eight Holstein heifers (457 ± 27 kg BW) were randomly assigned to a split-plot 4 × 4 Latin square experimental design with 18-d periods (14 d of adaptation and 4 d of sampling). Heifers were offered ad libitum and restricted intakes (85% of ad libitum intake) of diets differing in forage quality (high, corn silage; low, grass hay) and fiber content (high, 48% NDF; low 40% NDF). In each period, spot urine samples were taken 4 h after feeding on d 14 while total urine samples were composited from total urine collected during 4 sampling days. Creatinine excretion was affected by dietary treatment with lower values in heifers fed restricted intakes (70.06 vs. 79.55 mmol/day; P < 0.05). However, when expressed per BW, it was similar among treatments and lower than in previous studies (25 vs. 28 mg/kg BW). Restricted intake resulted in higher urine output and lower PD excretion (P < 0.05) regardless if estimated from spot sample (14.22 vs. 8.14 kg for urine output and 116.9 vs. 144.6 mmol/day for PD excretion) or observed in total urine collection (16.01 kg vs. 9.61 kg for urine output and 146.4 vs. 171.2 mmol/day for PD excretion). However, relationship between estimated and observed values for urine output was linear regardless of feeding regimen (R² = 0.75, RMSE = 2.73, mean bias = 5.22%) while the relationship for PD excretion was linear only for ad libitum intake (R² = 0.53, RMSE = 14.83, mean bias = 18.34% vs. R² = 0.01, RMSE = 24.35, mean bias = 29.34% for restricted). Spot urinary sampling could be used for daily urine output estimation in both ad libitum and restricted feeding regimens while it is suitable for estimation of PD excretion only in ad libitum intake.