| Summary: | The human metabolome may vary based on age, over time, and in the presence of viral carriage and bacterial colonization—a common scenario in children. We used nuclear magnetic resonance spectroscopy to identify and quantify urinary metabolites of children without signs or symptoms of respiratory illness. A urine sample and two nasopharyngeal swabs were collected to test for respiratory viral pathogens and colonization by <i>Streptococcus pneumoniae</i> (<i>Sp</i>). Urine samples were collected at the initial visit, 24 h post-enrollment, and 10–14 days post-enrollment. Of the 122 children enrolled, 24% had a virus detected and 19.7% had <i>Sp</i> detected. Intraclass correlation coefficients demonstrated greater within-subject versus between-subject variability for all metabolites detected. In linear mixed models adjusted for age, time, history of asthma, <i>Sp</i>, and viruses, 1-methylnicotinamide was increased by 50% in children with <i>Sp</i> and decreased by 35% in children with rhinovirus/enterovirus. Children with <i>Sp</i> had 83% higher levels of trimethylamine-N-oxide compared with those without <i>Sp</i>. However, when adjusting for multiple comparisons, the association was no longer statistically significant. In conclusion, there appear to be short-term changes within the urinary metabolome of healthy children, but levels of metabolites did not statistically differ in children with viral carriage or <i>Sp</i> detected.
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