The main purpose of the study was to analyze secular trends of health-related physical fitness in 7-14-year-old Croatian children and adolescents from 1999 and 2014. In this observational cross-sectional study, we recruited 5077 children and adolescents between ages 11 and 14 (50.8% girls) from five primary schools located in the capital city of Zagreb. Physical fitness performance was tested from 1999 until 2014. Physical fitness performance included: (1) body-mass index (measure of body size), (2) standing broad jump (measure of lower-body power), (3) polygon backwards (measure of general coordination and agility), (4) sit-ups in 60 s (measure of upper-body strength), (5) sit-and-reach test (measure of flexibility) and (6) 6-min run test (measure of cardiorespiratory fitness). Boys performed better in all physical fitness tests, except for sit-and-reach test (p < 0.001). In boys, between 1999 and 2014, body size, upper-body strength and coordination/agility increased, while flexibility, lower-body power and cardiorespiratory fitness decreased. During the same period, girls experienced an increase in body size, lower-body power, upper-body strength, coordination/agility and flexibility, while cardiorespiratory fitness decreased. This study shows that cardiorespiratory fitness, flexibility and coordination/agility decrease, while upper-body strength increases in both sexes. These findings should serve as an avenue for national monitoring system to screen and track biological development in children and adolescents.